KR20230009444A - Gene therapy by dysferin double vector - Google Patents

Abstract

Described herein are recombinant polynucleotides encoding fragments of the human dysferlin protein. Also further described are plasmids, viral vectors, dual vector systems, cells, and compositions comprising such recombinant polynucleotides. Such recombinant polynucleotides, plasmids, viral vectors, dual vector systems, cells, and compositions can be used to treat dysferinopathy.

Description

Gene therapy by dysferin double vector

This application claims priority under 35 U.S.C. §119(e) of US Provisional Application Serial No. 63/024,338, filed May 13, 2020, the contents of which are incorporated herein by reference.

Inclusion by reference of electronically submitted material

This application is a separate part of this disclosure, which is incorporated by reference in its entirety and has the following filename: 106887_8070_SL.txt; Size: 129,123 bytes; Creation Date: May 11, 2021 Contains a listing of sequences in computer readable form identified as such.

The present disclosure provides human dystrophy for treating subjects with dysferrin deficiency, e.g., limb girdle muscular dystrophy type 2B, miyoshi myopathy, and distal anterior compartment myopathy. Provided are polynucleotides comprising fragments of the dysferlin gene and plasmids, viral vectors, cells, and compositions comprising such polynucleotides, and methods of using the polynucleotides, plasmids, viral vectors, and compositions.

Dysferlinopathies are autosomal recessive disorders including limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal anterior compartment myopathy (collectively known as dysferlinopathies). Limb muscular dystrophy type 2B (LGMD2B) represents one of the most common LGMD in the United States, with worldwide reports of an incidence of 1/100,000-1/200,000. Miyoshi Myopathy is a more limited distal lower extremity form of dysferlinopathy. Indeed, given the disease spectrum, LGMD2B often begins distally with gastrocnemius atrophy and then spreads over time to affect the proximal muscles. Loss of dysferin results in a progressive form of dystrophy with chronic muscle fiber loss, inflammation, fat replacement and fibrosis, all of which exacerbate muscle weakness.

The dysferrin gene is large, with 55 exons identified so far spanning at least 150 kb of genomic DNA. These exons predict a cDNA of approximately 6.5 kb and a protein of 2,088 amino acids. Dysferin is a 237 kDa protein composed of a C-terminal hydrophobic transmembrane domain and a longer cytoplasmically oriented hydrophilic region with multiple C2 domains. Studies in the growing body have shown that loss of ^{dysperin impairs Ca 2+ -dependent} membrane repair in skeletal muscle [Song et al., Proc. Natl. Acad. Sci USA 98: 4084-4088, 2001; "Schnepp et al., J. Virol. 77:3495-3504, 2003]. Dysferin has also been shown to interact with other proteins involved in membrane repair, including annexins A1 and A2, AHNAK, and caveolin-3. The importance of these systems is emphasized given that skeletal muscle is mechanically active and prone to damage; Thus, a robust membrane resealing mechanism must exist. Absence or mutation of dysferin leads to a cascade of events that begins with damaged membrane repair and myofibrillar necrosis, leading to myofibrillar loss and progressive limb weakness. Loss of muscle fiber regeneration capacity is thought to be a contributing result of dysferrin deficiency. Dysferin is also involved in vesicle trafficking, endocytosis, and T tubule formation.

Mutations in the dysferin gene cause allelic autosomal recessive disorders including limb muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy and distal anterior compartment myopathy (collectively known as dysperrinopathies) [see, e.g., "Grose et al., PloS one 7:e39233,2012"; "Bansal et al., Nature 423, 168-172, 2003, Moore"; "Moore, SA, et al., J Neuropathol Exp. Neurol 65: 995-1003, 2006; "Rosales et al., Muscle Nerv 42:14-21, 2010"; "Sondergaard et al., Anns of Clin Trans Neurol 2:256-270, 2015"; "Evesson et al., J Biol Chem 285: 28529-28539, 2010"; and "Klinge et al., Soc Exp Biol 21: 1768-1776, 2007", each of which is incorporated by reference in its entirety]. A less common phenotype of dysferin deficiency is cervical syndrome (see Klinge et al., Muscle Nerve 41: 166-173, 2010, incorporated by reference in its entirety). Typically, patients present with slowly progressive decline and high serum creatine kinase (CK) in their early twenties. Approximately one-third of patients become wheelchair-dependent within 15 years of onset. Clinically, the heart is protected and cognitive function is unaffected. A phenotypic variant with a relatively limited distribution of muscle weakness sets the stage for potential site gene replacement therapy that could significantly impact quality of life for this disorder [Grose et al., PLoS One 7:e39233, 2012] , "Barton et al., Muscle Nerve 42:22-29, 2010"]. Single nucleotide changes are typical DYSF gene mutations, which favor success in gene transfer, which also serves to protect the transgene product from immune rejection [Rodine-Klapac et al., Mol Ther 18: 109-117, 2010 ”, “Mendell et al., N Eng J Med 363: 1429-1437, 2010”, “Mendell et al., Ann Neurol 66:290-297, 2009”, each of which is incorporated by reference in its entirety).

There is no cure or cure for dysperrinopathies. Overall, preclinical studies evaluating gene replacement or surrogate gene replacement have shown that a number of strategies show some efficacy in restoring membrane repair. The dysferrin gene contains 55 exons covering 150 kb of genomic DNA with its associated cDNA at 6.5 kb. However, for gene replacement, AAV's packaging limit is 4.7 kb, which is less than the cDNA sequence of dysferrin at 6.5 kb. Therefore, there is a need for treatments for LGMD2B that require new mechanisms to deliver functional full-length dysferrin protein to subjects in need thereof.

Disclosed herein is a recombinant polynucleotide encoding a fragment of a human dysferlin (hDYSF) protein, wherein the recombinant polynucleotide comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises: (a) SEQ ID NO: a nucleotide sequence of 1, 6, or 18; (b) at least 90%, 91%, 92% of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 18 over their respective full length of SEQ ID NO: 1, 6, or 18; 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; (c) the nucleotide sequence of SEQ ID NO: 13 or 15; (d) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; , 98%, or 99% identical nucleotide sequences; (e) a nucleotide sequence encoding a fragment of the hDYSF protein, the fragment consisting of the amino acid sequence of SEQ ID NO: 9; or (f) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of (e) over the entire length of the nucleotide sequence of (e), 98%, or 99% identical nucleotide sequences;

In some embodiments, the recombinant polynucleotide is derived from an inverted terminal repeat (ITR), promoter, intron, selection marker, or origin of replication (ORI). It further comprises one or more additional nucleotide sequences selected.

In some embodiments, the recombinant polynucleotide further comprises an additional nucleotide sequence comprising an ITR. In some embodiments, the ITR is an AAV ITR. In some embodiments, the AAV ITR is an AAV2 ITR or AAV3 ITR. In some embodiments, a recombinant polynucleotide comprises two ITRs. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 17.

In some embodiments, the recombinant polynucleotide further comprises an additional nucleotide sequence comprising a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is a human skeletal actin gene element, a cardiac actin gene element, a desmin promoter, a skeletal alpha-actin (ASKA) promoter, a troponin I (TNNI2) promoter, a myocyte-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid a-myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK7) promoter, C5-12 promoter, murine Creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene urea, hypoxia-inducible nuclear factor. In some embodiments, the muscle-specific promoter is the MHCK7 promoter. In some embodiments, the promoter is a recombinant promoter. In some embodiments, the recombinant promoter is a recombinant muscle-specific promoter. In some embodiments, the recombinant muscle-specific promoter is a recombinant myosin heavy chain-creatine kinase muscle-specific promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:4.

In some embodiments, the recombinant polynucleotide further comprises additional nucleotide sequences comprising introns. In some embodiments, an intron comprises a 5' donor site, branch point, and/or 3' splice site. In some embodiments, an intron is a chimeric intron. In some embodiments, the intron comprises a 5' donor region from the human β-globin gene. In some embodiments, an intron comprises a branch point from an immunoglobulin G (IgG) heavy chain. In some embodiments, an intron comprises a 3' splice acceptor site from an immunoglobulin G (IgG) heavy chain. In some embodiments, an intron comprises the nucleotide sequence of SEQ ID NO:5.

In some embodiments, the recombinant polynucleotide further comprises an additional nucleotide sequence comprising a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene is a β-lactamase gene or a kanamycin resistance gene. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO:18. In some embodiments, the recombinant nucleotide further does not include a second polynucleotide sequence encoding a second fragment of hDYSF protein.

In some embodiments, the recombinant nucleotides do not contain AAV sequences other than one or more ITRs.

In some embodiments, the recombinant nucleotides do not contain viral sequences other than one or more ITRs.

Disclosed herein is a recombinant polynucleotide sequence encoding a fragment of human dysferrin protein, wherein the recombinant polynucleotide comprises a second nucleotide sequence, wherein the second nucleotide sequence comprises: (a) SEQ ID NO: 2, 8, or nucleotide sequence of 19; (b) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (c) the nucleotide sequence of SEQ ID NO: 14 or 16; (d) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (e) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (f) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (e) over the entire length of the nucleotide sequence of (d); 98%, or 99% identical polynucleotide sequences;

In some embodiments, the recombinant polynucleotide comprises an inverted terminal repeat (ITR), selectable marker, origin of replication (ORI), untranslated region (UTR), or polyadenylation (polyA) signal. It further includes one or more additional nucleotide sequences that

In some embodiments, the recombinant polynucleotide further comprises an additional nucleotide sequence comprising an ITR. In some embodiments, the ITR is an AAV ITR. In some embodiments, the AAV ITR is an AAV2 ITR or AAV3 ITR. In some embodiments, a recombinant nucleotide comprises two ITRs. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 3 or 17.

In some embodiments, the recombinant polynucleotide further comprises a nucleotide sequence comprising a polyA signal. In some embodiments, the polyA signal is an artificial polyA signal. In some embodiments, the polyA signal comprises the nucleotide sequence of SEQ ID NO:7.

In some embodiments, the recombinant polynucleotide further comprises an additional nucleotide sequence comprising a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene is a β-lactamase gene or a kanamycin resistance gene. In some embodiments, the recombinant polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 19.

In some embodiments, the recombinant nucleotide further does not comprise a first polynucleotide sequence encoding a first fragment of hDYSF protein.

In some embodiments, the recombinant nucleotides do not contain AAV sequences other than one or more ITRs.

In some embodiments, the recombinant nucleotides do not contain viral sequences other than one or more ITRs.

Further disclosed herein is a dual adeno-associated viral (AAV) vector system comprising:

(a) a first AAV vector, wherein the first AAV vector comprises a first recombinant polynucleotide encoding an N-terminal fragment of human dysferrin (hDYSF) protein, wherein the first recombinant polynucleotide comprises a first nucleotide sequence wherein the first nucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 1, 6, or 18; (ii) at least 90%, 91%, 92%, 93%, 94% of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 18 over their respective full length of SEQ ID NO: 1, 6, or 18; , 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; 98%, or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of the hDYSF protein, said fragment consisting of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) a second AAV vector, wherein the second AAV vector comprises a second recombinant polynucleotide encoding a C-terminal fragment of human dysferrin protein, wherein the second recombinant polynucleotide comprises a second nucleotide sequence; , wherein the second nucleotide sequence is: (i) the nucleotide sequence of SEQ ID NO: 2, 8, or 19; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 98%, or 99% identical polynucleotide sequences;

includes

Further disclosed herein are adeno-associated viral (AAV) vectors comprising any of the recombinant polynucleotides disclosed herein. In some embodiments, the recombinant polynucleotide encodes an N-terminal fragment of human dysferrin protein. In some embodiments, the recombinant polynucleotide encodes a C-terminal fragment of human dysferrin protein. In some embodiments, the AAV vector is AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV- 11, AAV-12, AAV-13, AAVrh10, AAVrh20, or AAVrh74. In some embodiments, the AAV vector is AAVrh74.

Further disclosed herein are compositions comprising any of the AAV vectors disclosed herein.

Further disclosed herein is a composition comprising:

(a) a first recombinant adeno-associated virus (rAAV) vector, wherein the first rAAV vector comprises a first recombinant polynucleotide encoding an N-terminal fragment of human dysferrin (hDYSF) protein, wherein the first recombinant The polynucleotide comprises a first nucleotide sequence comprising: (i) the nucleotide sequence of SEQ ID NO: 1, 6, or 18; (ii) at least 90%, 91%, 92%, 93%, 94% of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 18 over their respective full length of SEQ ID NO: 1, 6, or 18; , 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; 98%, or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of the hDYSF protein, said fragment consisting of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) a second rAAV vector, wherein the second rAAV vector comprises a second recombinant polynucleotide encoding a C-terminal fragment of human dysferrin protein, wherein the second recombinant polynucleotide comprises a second nucleotide sequence; , wherein the second nucleotide sequence is: (i) the nucleotide sequence of SEQ ID NO: 2, 8, or 19; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 98%, or 99% identical polynucleotide sequences;

includes

In some embodiments, the molar ratio of the first and second rAAV vectors is about 100:1-100, about 10:1-1:10, about 2:1-1:2, or about 1:1.

Further disclosed herein is an Adeno-associated virus (AAV) vector, wherein the AAV vector:

(a) a first inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the polynucleotide comprises (i) a nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(c) a second ITR;

Including, wherein the polynucleotide is flanked (or 'next to') by the first and second ITRs.

In some embodiments, the ITR is an AAV ITR. In some embodiments, the AAV ITR is an AAV2 ITR or AAV3 ITR. In some embodiments, the first and/or second ITR comprises the nucleotide sequence of SEQ ID NO: 3 or 17.

In some embodiments, the AAV vector further comprises one or more additional polynucleotide sequences comprising a promoter, intron, selectable marker, or origin of replication (ORI).

In some embodiments, the AAV vector further comprises an additional polynucleotide sequence comprising a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is a myosin heavy chain complex-E box muscle creatine kinase fusion enhancer/promoter. In some embodiments, the promoter is a recombinant promoter. In some embodiments, the recombinant promoter is a recombinant muscle-specific promoter. In some embodiments, the recombinant muscle-specific promoter is the MHCK7 promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:4.

In some embodiments, the AAV vector further comprises additional polynucleotide sequences comprising introns. In some embodiments, an intron comprises a 5' donor site, branch point, and/or 3' splice site. In some embodiments, an intron is a chimeric intron. In some embodiments, the intron comprises a 5' donor region from the human β-globin gene. In some embodiments, an intron comprises a branch point from an immunoglobulin G (IgG) heavy chain. In some embodiments, an intron comprises a 3' splice acceptor site from an immunoglobulin G (IgG) heavy chain. In some embodiments, an intron comprises the nucleotide sequence of SEQ ID NO:5.

In some embodiments, the AAV vector further comprises an additional polynucleotide sequence comprising a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene is a β-lactamase gene or a kanamycin resistance gene. In some embodiments, the AAV vector comprises the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO:15.

In some embodiments, the AAV vector does not further comprise a second polynucleotide sequence encoding a second fragment of hDYSF protein.

In some embodiments, the AAV vector does not contain AAV sequences other than one or more ITRs.

In some embodiments, the AAV vector does not contain viral sequences other than one or more ITRs.

Further disclosed herein is an adeno-associated viral (AAV) vector, wherein the adeno-associated viral (AAV) vector:

(a) a first inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin protein, wherein the polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2 or 8; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; or 99% identical nucleotide sequences; (v) a polynucleotide encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 98%, or 99% identical polynucleotides; and

(c) a second ITR;

Including, wherein the polynucleotide is flanked by the first and second ITRs.

In some embodiments, the AAV vector further comprises one or more polynucleotide sequences comprising a selectable marker, origin of replication (ORI), untranslated region (UTR), or polyadenylation (polyA) signal.

In some embodiments, the ITR is an AAV ITR. In some embodiments, the AAV ITR is an AAV2 ITR or AAV3 ITR. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 3 or 17.

In some embodiments, the AAV vector further comprises an additional polynucleotide sequence comprising a polyA signal. In some embodiments, the polyA signal is an artificial polyA signal. In some embodiments, the polyA signal comprises the nucleotide sequence of SEQ ID NO:7.

In some embodiments, the AAV vector further comprises an additional polynucleotide sequence comprising a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene is a β-lactamase gene or a kanamycin resistance gene.

In some embodiments, the AAV vector comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

In some embodiments, the AAV vector does not further comprise a second polynucleotide sequence encoding a second fragment of hDYSF protein.

In some embodiments, the AAV vector does not contain AAV sequences other than one or more ITRs.

In some embodiments, the AAV vector does not contain viral sequences other than one or more ITRs.

Disclosed herein is a dual adeno-associated virus (AAV) vector system, wherein the dual AAV vector system:

(I) a first AAV vector, wherein the first AAV vector comprises:

(a) a first AAV vector comprising a first inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the polynucleotide comprises (i) a nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(c) a second ITR;

Including, wherein the polynucleotide is flanked by the first ITR and the second ITR; and

(II) a second AAV vector, wherein the second AAV vector,

(a) a second AAV vector comprising a third inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin protein, wherein the polynucleotide comprises: (i) a nucleotide sequence of SEQ ID NO: 2 or 8; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; or 99% identical nucleotide sequences; (v) a polynucleotide encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); are 98%, or 99% identical polynucleotides; and

(c) a fourth ITR;

wherein the polynucleotide is flanked by a third ITR and a fourth ITR;

includes

Further disclosed herein is an adeno-associated virus (AAV) packaging system comprising: (a) a plasmid comprising a recombinant polynucleotide disclosed herein; (b) an adenovirus helper plasmid; and (c) rep-cap plasmid; In some cases, the adenoviral helper plasmid includes a pHELP plasmid.

Further provided herein are: (a) a plasmid comprising a recombinant polynucleotide; and (b) an adenovirus helper plasmid. In some cases, the adenoviral helper plasmid includes a pHELP plasmid.

Further disclosed herein is a method of producing an adeno-associated virus (AAV) vector comprising contacting a cell with an AAV packaging system, wherein the AAV packaging system comprises (a) a recombinant polynucleotide disclosed herein. a plasmid; (b) an adenovirus helper plasmid; and (c) rep-cap plasmid; In some cases, the cell is a host cell, optionally a mammalian host cell, and further optionally HEK293.

Further disclosed herein is a method of producing an adeno-associated virus (AAV) vector comprising transducing a packaging cell line with an AAV packaging system, the AAV packaging system comprising: (a) the disclosed herein a plasmid comprising an AAV expression cassette comprising any of the recombinant polynucleotides; and (b) an adenovirus helper plasmid; wherein the packaging cell line expresses adeno-associated virus rep and cap genes. In some cases, the AAV rep gene is Rep78. In some cases, the AAV cap gene is the Rh74 cap gene.

Further disclosed herein are cells comprising any of the recombinant polynucleotides disclosed herein.

Further disclosed herein are cells comprising an AAV expression cassette comprising any of the recombinant polynucleotides disclosed herein. In some cases, the plasmid comprises a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18 or 19. In some cases, the plasmid comprises a polynucleotide of SEQ ID NO: 18 or 19.

Further disclosed herein is a method of treating dysferinopathy, the method comprising:

(a) an effective amount of a first recombinant polynucleotide comprising a first polynucleotide sequence encoding the N-terminus of human dysferrin (hDYSF) protein, wherein said first polynucleotide sequence comprises (i) SEQ ID NOs: 1, 6 , or a nucleotide sequence of 18; (ii) at least 90%, 91%, 92%, 93%, 94 to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 18 over their respective full length of SEQ ID NO: 1, 6, or 18; %, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; , 98%, or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of the hDYSF protein, wherein the fragment of the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v), 98%, or 99% identical nucleotide sequences; and

(b) an effective amount of a second recombinant polynucleotide comprising a second polynucleotide sequence encoding a C-terminal fragment of human dysferrin protein, wherein the second polynucleotide sequence comprises: (i) SEQ ID NO: 2, 8, or nucleotide sequence of 19; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; , 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% relative to the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; , 98%, or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). , 98%, or 99% identical polynucleotide sequences;

It includes administering

In some embodiments, the first polynucleotide is administered intramuscularly or intravenously. In some embodiments, the second polynucleotide is administered intramuscularly or intravenously.

In some embodiments, the first and second polynucleotides are administered simultaneously or sequentially.

In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

In addition, in the present application, to a subject in need of treatment for dysperinopathies,

(a) an effective amount of a first adeno-associated virus (AAV) vector, wherein the first AAV vector comprises a first polynucleotide encoding the N-terminus of a human dysferrin (hDYSF) protein, wherein the first polynucleotide is, (i) the nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of the hDYSF protein, wherein the fragment of the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) an effective amount of a second AAV vector, wherein the second AAV vector comprises a second polynucleotide encoding a C-terminal fragment of human dysferrin protein, wherein the second polynucleotide comprises: (i) SEQ ID NO: 2 or a nucleotide sequence of 8; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; or 99% identical nucleotide sequences; (v) a polynucleotide encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the polynucleotide of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical polynucleotides;

A method of treating dysperinopathies, comprising administering a, is disclosed.

In some embodiments, the first AAV vector is administered intramuscularly or intravenously. In some embodiments, the second AAV vector is administered intramuscularly or intravenously. In some embodiments, the first and second AAV vectors are administered simultaneously.

In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

In some embodiments, disclosed herein are methods of treating dysferinosis comprising administering to a subject in need thereof an effective amount of any of the AAV dual vector systems disclosed herein.

In some embodiments, the AAV dual vector system is administered intramuscularly or intravenously.

In some embodiments, the dysferinosis is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

In some embodiments, disclosed herein are methods of treating dysferinosis comprising administering to a subject in need thereof an effective amount of any of the compositions disclosed herein.

In some embodiments, the composition is administered intramuscularly or intravenously.

In some embodiments, the dysferinosis is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

Further disclosed herein is the use of the composition in the manufacture of a medicament for treating dysferinopathy in a subject in need thereof, wherein the composition comprises:

(a) a first recombinant polynucleotide comprising a first polynucleotide sequence encoding the N-terminus of a human dysferrin (hDYSF) protein, wherein the first polynucleotide sequence is: (i) SEQ ID NO: 1, 6, or a nucleotide sequence of 18; (ii) at least 90%, 91%, 92%, 93%, 94% of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 18 over their respective full length of SEQ ID NO: 1, 6, or 18; , 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; 98%, or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) a second recombinant polynucleotide comprising a second polynucleotide sequence encoding a C-terminal fragment of human dysferrin protein, wherein said second polynucleotide sequence is: (i) of SEQ ID NO: 2, 8, or 19 nucleotide sequence; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 98%, or 99% identical polynucleotide sequences;

includes

In some embodiments, the first polynucleotide is administered intramuscularly or intravenously. In some embodiments, the second polynucleotide is administered intramuscularly or intravenously. In some embodiments, the first and second polynucleotides are administered simultaneously.

In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

Further disclosed herein is the use of the composition in the manufacture of a medicament for treating dysferinopathy in a subject in need thereof, wherein the composition comprises:

(a) an effective amount of a first adeno-associated virus (AAV) vector, wherein the first AAV vector comprises a first polynucleotide sequence encoding the N-terminus of a human dysferrin (hDYSF) protein, wherein the first polynucleotide sequence The nucleotides include (i) the nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) an effective amount of a second adeno-associated virus (AAV) vector, wherein the second AAV vector comprises a second polynucleotide encoding a C-terminal fragment of human dysferrin protein, wherein the second polynucleotide comprises: (i) the nucleotide sequence of SEQ ID NO: 2 or 8; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; or 99% identical nucleotide sequences; (v) a polynucleotide encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the polynucleotide of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical polynucleotides;

includes

In some embodiments, the first AAV vector is administered intramuscularly or intravenously. In some embodiments, the second AAV vector is administered intramuscularly or intravenously. In some embodiments, the first and second AAV vectors are administered simultaneously.

In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

Disclosed herein is the use of a composition in the manufacture of a medicament for treating dysferinopathy in a subject in need thereof, wherein the composition comprises any AAV dual vector system disclosed herein. .

In some embodiments, the composition is administered intramuscularly or intravenously.

In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

Disclosed herein is the use of any disclosed composition in the manufacture of a medicament for the treatment of dysferinopathy in a subject in need thereof.

In some embodiments, the composition is administered intramuscularly or intravenously.

In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

In some embodiments, the effective amount of the first AAV vector is about 1×10 ⁶ to 1×10 ¹⁶ vg/kg, about 1×10 ⁸ to 1×10 ¹⁵ vg/kg, based on supercoil DNA or plasmid as a quantification standard. kg, or about 1×10 ¹⁰ to 1×10 ¹⁴ vg/kg.

In some embodiments, the effective amount of the second AAV vector is about 1×10 ⁶ to 1×10 ¹⁶ vg/kg, about 1×10 ⁸ to 1×10 ¹⁵ vg/kg, based on supercoil DNA or plasmid as a quantification standard. kg, or about 1×10 ¹⁰ to 1×10 ¹⁴ vg/kg.

In some embodiments, the first AAV vector is administered at least 1, 2, 3, 4, or 5 times.

In some embodiments, the second AAV vector is administered at least 1, 2, 3, 4, or 5 times.

In some embodiments, the effective amount of the AAV dual vector system is about 1×10 ¹⁰ to 1×10 ¹³ vector genome (vg), about 1×10 ¹¹ to 1×10 ¹³ vg, 1×10 ¹² to 1×10 ¹³ vg. to be.

In some embodiments, the AAV dual vector system is administered at least 1, 2, 3, 4, or 5 times.

In some embodiments, the effective amount of the composition is about 1×10 ¹⁰ to 1×10 ¹³ vector genome (vg), about 1×10 ¹¹ to 1×10 ¹³ vg, 1×10 ¹² to 1×10 ¹³ vg.

In some embodiments, the composition is administered at least 1, 2, 3, 4, or 5 times.

Disclosed herein is a recombinant polynucleotide encoding a human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences. In some embodiments, the recombinant polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 20.

Disclosed herein is a method for preparing a recombinant polynucleotide encoding human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide sequence is at least 90%, 91% of the nucleotide sequence of SEQ ID NO: 20 or the nucleotide sequence of SEQ ID NO: 20 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, the method comprising:

A recombinant polynucleotide comprising a first polynucleotide sequence encoding the N-terminus of a human dysferrin (hDYSF) protein, wherein the first polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 1, 6, or 18; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 1, 6, or 18 over the entire length of each of SEQ ID NO: 1, 6, or 18; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; 98%, or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) a second recombinant polynucleotide comprising a second polynucleotide sequence encoding a C-terminal fragment of human dysferrin protein, wherein said second polynucleotide sequence is: (i) of SEQ ID NO: 2, 8, or 19 nucleotide sequence; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 98%, or 99% identical polynucleotide sequences;

including contact with

Disclosed herein is a method for producing a recombinant polynucleotide encoding a human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, the method comprising:

(I) a first AAV vector, wherein the first AAV vector comprises:

(a) a first inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the polynucleotide comprises (i) a nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(c) a second ITR;

wherein said polynucleotide is flanked by said first and second ITRs, said second AAV vector comprising said (a) a first inverted terminal repeat (ITR); and

(II) a second AAV vector, wherein the second AAV vector,

(a) a third inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin protein, wherein the polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2 or 8; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the polynucleotide of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical polynucleotide sequences; consisting of; and

(c) a fourth ITR;

Including, wherein the polynucleotide is flanked by the third and fourth ITRs;

Including contacting with a dual AAV vector system, including.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cells are muscle cells, heart cells, stem cells, satellite cells, and/or liver cells.

Disclosed herein is a method for preparing a recombinant polynucleotide encoding human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide sequence is at least 90%, 91% of the nucleotide sequence of SEQ ID NO: 20 or the nucleotide sequence of SEQ ID NO: 20 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, wherein the method comprises:

A first polynucleotide sequence encoding the N-terminus of a human dysferrin (hDYSF) protein, wherein the first polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 1, 6, or 18; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 1, 6, or 18 over the entire length of each of SEQ ID NO: 1, 6, or 18; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; 98%, or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) a second polynucleotide sequence encoding a C-terminal fragment of human dysferrin protein, wherein the second polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2, 8, or 19; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); consisting of polynucleotide sequences that are 98%, or 99% identical;

It includes administering a recombinant polynucleotide comprising a.

Disclosed herein is a method for producing a recombinant polynucleotide encoding a human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, the method comprising:

(I) a first AAV vector, wherein the first AAV vector comprises:

(a) a first inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the polynucleotide comprises (i) a nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(c) a second ITR;

wherein said polynucleotide is flanked by said first and second ITRs, said second AAV vector comprising said (a) a first inverted terminal repeat (ITR); and

(II) a second AAV vector, wherein the second AAV vector,

(a) a third inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin protein, wherein the polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2 or 8; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 98%, or 99% identical polynucleotide sequences; and

(c) a fourth ITR;

Including, wherein the polynucleotide is flanked by the third and fourth ITRs;

It includes administering a dual AAV vector system, comprising.

Disclosed herein is a method of treating muscular dystrophy in a subject comprising expression of a recombinant polynucleotide encoding human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide sequence is the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 20 in a subject a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of 20.

In some embodiments, the method provides a subject with

A first polynucleotide sequence encoding the N-terminus of a human dysferrin (hDYSF) protein, wherein the first polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 1, 6, or 18; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 1, 6, or 18 over the entire length of each of SEQ ID NO: 1, 6, or 18; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; 98%, or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(b) a second polynucleotide sequence encoding a C-terminal fragment of human dysferrin protein, wherein the second polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2, 8, or 19; (ii) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (v) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); consisting of polynucleotide sequences that are 98%, or 99% identical;

It includes administering a recombinant polynucleotide comprising a.

In some embodiments, the method comprises:

(I) a first AAV vector, wherein the first AAV vector comprises:

(a) a first inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the polynucleotide comprises (i) a nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; or 99% identical nucleotide sequences; (v) a nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, or 99% identical nucleotide sequences; and

(c) a second ITR;

wherein said polynucleotide is flanked by said first and second ITRs, said second AAV vector comprising said (a) a first inverted terminal repeat (ITR); and

(II) a second AAV vector, wherein the second AAV vector,

(a) a third inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin protein, wherein the polynucleotide comprises: (i) a nucleotide sequence of SEQ ID NO: 2 or 8; (ii) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; or 99% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; or 99% identical nucleotide sequences; (v) a polynucleotide encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 98%, or 99% identical polynucleotide sequences; and

(c) a fourth ITR;

Including, wherein the polynucleotide is flanked by the third and fourth ITRs;

It includes administering a dual AAV vector system, comprising.

In some embodiments, the subject is a human, non-human primate, canine, ovine, horse, porcine, murine, rat, rabbit, cow (bovine), or a mammal selected from feline.

In some embodiments, the subject suffers from dysferlinopathy. In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

1 provides a schematic diagram of a dual AAV vector system for treating dysferinopathies. The 5' vector (e.g. 5' hDYSF AAV vector), pAAV.MHCK7.DYSF5'.PTG (PTG=promoter/transgene) contains the muscle specific MHCK7 promoter, chimeric intron, consensus Kozak sequence and It contains the 5' portion of DYSF cDNA corresponding to amino acids 1-1113 of SEQ ID NO: 12. A 3' vector (e.g., a 3' hDYSF AAV vector), pAAV.DYSF3'.POLYA, is a DYSF 3'UTR containing the 3' portion of the DYSF cDNA corresponding to amino acids 794-2080 of SEQ ID NO: 12 and a polyadenylation signal. contains
Figure 2 provides the pAAV.MHCK7.DYSF5'.PTG DNA vector plasmid map.
Figure 3 provides the pAAV.DYSF3'.POLYA vector DNA plasmid map.
4A-4D show dysferrin expression after delivery of the dual vector system. Robust full-length dysferlin expression was shown after delivery of both vectors by immunostaining (Fig. 4a) and western blot (Fig. 4c). Delivery of either vector alone had no aberrant dysferrin expression (FIG. 4B: immunostaining, FIG. 4D: Western blot). 3222 is full-length control.
5A to 5C show the results of the time course of dysferin expression after rAAVrh.74.MHCK7.DYSF.DV delivery. 5A demonstrates full-length dysferrin expression by dysferrin immunolabeling (upper panel) seen after delivery of dual vectors into the left tibialis anterior (LTA). Dysferin expression persisted for 1, 3, and 6 months after treatment with no abnormal response in pathology (H&E, lower panel). Scale bar, 100 μm. N=4 per time point. 5B shows Western blots for 1, 3, and 6 month samples demonstrating expression of full-length dysferrin in injected LTA (n=2 per group). 5C depicts biodistribution plots of genomic DNA per μg vector genome at 3 and 6 months post injection for various tissues. Note: LTA was treated; log axis.
6 Western blot analysis of target muscle (LTA) and non-target tissues from 4 separate animals treated by intramuscular injection with rAAVrh.74.MHCK7.DYSF.DV at endpoints of 3 or 12 months. indicates
7A-7C show dysferrin expression after systemic delivery of AAVrh.74.MHCK7.DYSF.DV. Figure 7a shows dysferrin immunolabeling of tissues after systemic delivery of 6×10 ¹² vg (2.4e13 vg/kg, based on supercoil DNA or plasmid as quantitative standard) AAVrh.74.MHCK7.DYSF.DV ( n=6/dose). Muscles shown are for cardiac, gastrocnemius, diaphragm and quadriceps, treated (AAV.DV) and wild type (WT) tissues. 7B shows quantification of centralized nuclei in the tibialis anterior (LTA), gastrocnemius (RGAS), quadriceps (LQD), triceps (RTri) and diaphragm. *p<0.05 significant difference between sample and wild type, # no significant difference between sample and wild type. Figure 7C is a Western image of tissue lysates (H: heart, G: gastrocnemius, Q: quadriceps, D: diaphragm) demonstrating a full-length dysperin band at 237 kD γ-tubulin included as a loading control. represents a blot.
Figure 8 shows dose-dependent membrane resealing activity after AAVrh.74.DYSF.DV delivery.
9 shows reversal of fibrosis and inflammation following systemic delivery of AAVrh.74.MHCK7.DYSF.DV. BlaJ mice were treated with 6×10 ¹² vg (2.4e13 vg/kg) based on supercoil DNA or plasmid as a quantification standard. AAVrh.74.MHCK7.DYSF.DV (n=6). Porcine muscles were removed and analyzed for fibrosis (middle column) and the presence of CD8 mononuclear cells. There was a significant decrease in both parameters after gene transfer.
10A-10B demonstrate systemic delivery of rAAVrh.74.MHCK7.DYSF.DV. Functional defects were restored in Dysf−/− mice. 10A: Diarrheal muscle strips were harvested and subjected to a protocol to evaluate specific forces. The treated diaphragm contained 2e12 vg total AAV.DYSF.DV (8e13 vg/kg based on supercoiled DNA or plasmid as quantification standard), or 6e12 vg total AAV.DYSF.DV (based on supercoiled DNA or plasmid as quantification standard). 2.4e13 vg/kg) demonstrated significant improvement in force (**P>0.01, ANOVA) not different from wild-type force at both doses. There was no significant improvement at the lower dose.
11A-11D show results from monitoring of T cell responses to AAV capsids and dysferrin. Peripheral blood mononuclear cells were isolated and exposed to peptides including AAV5 and AAVrh.74 capsids (blue bars) as well as human dysferrin (green). T cell responses to AAV5 capsid and dysferrin were monitored at 3 months (FIG. 11A) and 6 months (FIG. 11B). T cell responses to AAVrh.74 capdis and dysferrin were monitored at 3 months (FIG. 11C) and 6 months (FIG. 11D).
12A-12C show dysferrin expression in non-human primates. 12A shows histology (H&E) and dysferrin immunofluorescence (IF) images of NHP tissue at 3 and 6 months after injection of AAV5.DYSF or AAVrh.74.DYSF.DV. H&E stained sections show immune infiltration of fibers and lack of necrosis. 12B shows Western blot images of tissues from 3 and 6 months post injection for both AAV5.DYSF and AAVrh.74.DYSF.DV. Importantly, the injected tissues demonstrate overexpression of dysferin compared to sham controls. The positive (+) control is wild type mouse tissue, and the negative (-) control is 129Dysf−/− non-injected tissue. 12C shows the biodistribution of the vector genome after IM injection with AAVrh.74.DYSF.DV into the left TA, note the logarithmic scale.
13 demonstrates the use of anti-FLAG to confirm vector derived dysferrin expression. An N-terminal FLAG tag was used to distinguish endogenous and AAV-derived dysferrin.

Justice

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Additionally, terms as defined in commonly used dictionaries are to be construed to have a meaning consistent with their meaning in the context of this application and related fields, and are not to be idealized or overly formal unless expressly so defined herein. It will be understood that it should not be interpreted in a meaningful way. Although not explicitly defined below, these terms should be interpreted according to their ordinary meaning.

The terminology used in the description herein is for the purpose of describing specific embodiments only and is not intended to limit the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

The practice of the present technology will utilize, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art.

Unless the context dictates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. For illustrative purposes, when the specification refers to a complex as comprising components A, B, and C, any of A, B, or C, or combinations thereof, singularly or in any combination, are omitted and identified as possible. specifically intended

Unless expressly indicated otherwise, all stated embodiments, features, and terms are intended to include both the recited embodiments, features, or terms and their biological equivalents.

All numerical designations, such as pH, temperature, time, concentration, and molecular weight, as appropriate, by increments of 1.0 or 0.1, or alternatively +/- 15%, or alternatively 10%, or alternatively 5%. %, or alternatively an approximation varying by a variance of 2%, such ranges are inclusive. Although not always explicitly stated, it should be understood that all numerical designations are preceded by the term “about”. Also, although not always explicitly stated, it is to be understood that the reagents described herein are merely illustrative, and such equivalents are known in the art.

The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. See, eg, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); "Current Protocols In Molecular Biology (F M Ausubel, et al., eds, (1987))"; 「Methods in the series Methods in Enzymology (Academic Press, Inc): PCR 2: A Practical Approach (M J MacPherson, B D Hames and G R Taylor eds (1995)), Harlow and Lane, eds (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R I Freshney, ed (1987)).

As used herein, the terms "increased", "reduced", "high", "low" or any grammatical variations thereof refer to about 90%, 80%, 50%, 20%, A variation of 10%, 5%, 1%, 0.5%, or even 0.1%.

The terms “acceptable,” “effective,” or “sufficient,” when used to describe a selection of any ingredient, range, dosage form, etc. disclosed herein, mean that the ingredient, range, dosage form, etc. is suitable for the disclosed purpose. it is intended

Also, as used herein, the term “and/or” refers to and encompasses any and all possible combinations of one or more associated listed items, as well as the lack of combinations (“or”) when alternatively interpreted.

Unless expressly cited and otherwise intended, when this disclosure is directed to polypeptides, proteins, polynucleotides or antibodies, equivalents or bioequivalents thereof are to be inferred to fall within the scope of this disclosure. As used herein, the term "biological equivalent thereof", when referring to a reference protein, antibody, polypeptide or nucleic acid, is intended to be synonymous with "equivalent thereof", and is intended to have minimal sequence identity while still retaining the desired structure or functionality. do. Unless specifically stated herein, any polynucleotide, polypeptide or protein mentioned herein is also contemplated to include equivalents thereof. For example, an equivalent may have at least about 70% homology or identity, or at least 80% homology or identity, and alternatively, at least about 85%, or alternatively, at least about 90%, or alternatively, at least about 90% homology or identity over the length of the reference sequence. Typically at least about 95%, or alternatively, 98% homology or identity is intended, and exhibits biological activity that is substantially equivalent to a reference protein, polypeptide or nucleic acid. Alternatively, when referring to a polynucleotide, an equivalent thereof is, in one aspect, a reference polynucleotide or its complement that has the same or similar activity or function as the reference polynucleotide or its complement, in a further aspect, under stringent conditions. It is a polynucleotide that hybridizes to the body.

Equivalents of proteins or polypeptides (referred to herein as references) share at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) identity to the reference, and the functions of the reference and maintain manufacturability.

As used herein, the terms "function", "activity", and "enzymatic activity" are used interchangeably. Loss of ^{dysferrin has been shown to impair Ca 2+ -dependent} membrane repair in skeletal muscle [Song et al., Proc Natl Acad Sci USA 98:4084-4088, 2001] and [Schnepp et al., J Virol 77:3495-3504 , 2003]. Dysferin has also been shown to interact with other proteins involved in membrane repair, including annexins A1 and A2, AHNAK, and caveolin-3 [Schnepp et al., J Virol 77:3495-3504, 2003] , "Duan et al., J Virol 72:8568-8577, 1998", "Donsante et al., Gene Ther 8:1343-1346, 2001", and "Monahan et al., Expert Opin Drug Saf 1:79-91, 2002"] . Loss of muscle fiber regeneration capacity is thought to be a contributing result of dysferrin deficiency (see Song et al., Gene Ther 8:1299-1306, 2001). Dysferin has also been implicated in vesicle trafficking and endocytosis and T tubule formation [Eveson et al., The Journal of Biological Chemistry 285:28529-28539, 2010], Klinge et al., FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 21:1768-1776, 2007, and Klinge et al., Muscle & Nerve 41:166-173, 2010]. Thus, examples of activities of dysferrin include, but are not limited to, membrane repair in skeletal muscle, e.g., membrane resealing, prevention or restoration of muscle fiber regenerative capacity, vesicle trafficking, endocytosis, and transverse (T-) tubule formation. Not limited. Membrane repair assays, vesicle trafficking assays, and tube formation assays are known in the art and can be used to measure dysferrin activity in vitro. See, eg, Carmeille et al., Methods Mol Biol 1668:195-207, 2017, Vassilieva and Nusrat, Methods Mol Biol 440:3-14, 2008, Demonbreun et al., Am J Pathol 184(1):248 -59, 2014”, each of which is incorporated by reference in its entirety. Additional methods for measuring the activity of dysferrin are found, for example, in Grose et al., PLoS One 7:e39233, 2012 and Sondergaard et al., Anns of Clin Trans Neurol 2:256-270, 2015. .

Equivalents of polynucleotides (referred to herein as references) share at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) identity to the reference, and are encoded by the reference. encodes the same polypeptide as described above, or encodes an equivalent of the polypeptide encoded by reference.

A sequence alignment is performed between the test sequence and the reference sequence to arrive at a position or contiguous segment of the test sequence that is equivalent to (or equivalent to) an amino acid/nucleotide residue or contiguous segment of the reference sequence. Positions or segments aligned with respect to each other are determined as equivalents.

The term "affinity tag" refers to a fusion protein to allow detection of the fusion protein and/or purification of the fusion protein from the cellular environment using a ligand capable of binding to the affinity tag, i.e., having affinity for the affinity tag. Refers to a polypeptide that can be included within. A ligand can be, but is not limited to, an antibody, resin, or complementary polypeptide. Affinity tags can include small peptides, typically about 4 to 16 amino acids in length, or they can include larger polypeptides. Commonly used affinity tags include polyarginine, FLAG, V5, polyhistidine, c-Myc, Strep II, maltose binding protein (MBP), N-utilizing substance protein A (NusA), thioredoxin (Trx), among others. ), and glutathione S-transferase (GST) (see, eg, "GST Gene Fusion System Handbook - Sigma-Aldrich"). In one embodiment, the affinity tag is a polyhistidine tag, such as a His6 tag (SEQ ID NO: 21). Inclusion of an affinity tag within the fusion protein allows the fusion protein to be purified from the cellular environment by affinity purification using an affinity medium capable of binding tightly and specifically to the affinity tag. The affinity medium may include, for example, a metal-filled resin or a ligand covalently linked to a stationary phase (matrix) such as agarose or metal beads. For example, polyhistidine-tagged fusion proteins (also referred to as His-tagged fusion proteins ^{) can be recovered by immobilized metal ion chromatography using Ni 2+ or Co 2+ loaded resins} , ^and A -FLAG affinity gel can be used to capture FLAG tagged fusion proteins, and glutathione cross-linked to a solid support such as agarose can be used to capture GST tagged fusion proteins. In one aspect, the affinity tag is a purification tag or marker.

As used herein, the term "purifying", "purifying" or "isolating" refers to one or more biomaterials (eg, polynucleotides, polypeptides or viral vectors) from a complex mixture, such as a cell lysate or mixture of polypeptides. Refers to the process of isolating. The purification, separation or isolation need not be complete, ie some components of the complex mixture may remain with one or more biomaterials (eg polynucleotides, polypeptides or viral vectors) after the purification process. However, the product of purification must be enriched for one or more biomaterials (e.g. polynucleotides, polypeptides or viral vectors) relative to the complex mixture prior to purification, and a significant fraction of the other components initially present in the complex mixture are purified. It must be removed by the process.

As used herein, the term “cell” may refer to a prokaryotic or eukaryotic cell, optionally obtained from a subject or from a commercially available source. In some cases, a cell is a host cell, eg, a mammalian cell or mammalian host cell. In some cases, host cells are also referred to herein as production cells or packaging cells. In some cases, the cell line is a packaging cell line.

A "nucleated cell" includes all living organisms except monera. They can be readily distinguished through their membrane-bound nuclei. Animals, plants, fungi and protists are eukaryotes or organisms in which cells are organized into complex structures by internal membranes and cytoskeletons. The most characteristic membrane-bound structure is the nucleus. Unless specifically stated otherwise, the term "host" includes eukaryotic hosts including, for example, yeast, higher plants, insects and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, pig, murine, rat, avian, reptile and human, e.g. HEK293 cells, Chinese Hamster Ovary (CHO) cells, CHO-S cells, CHO-K1 cells, 293T cells, HeLa cells, barbie hamster kidney (BHK) cells, Sf9 cells, stem cells, satellite cells and muscle cells. Examples of muscle cells include, but are not limited to, skeletal muscle cells, cardiac muscle cells, and smooth muscle cells.

A "nucleated cell" that usually lacks a nucleus or any other membrane-bound organelle and divides into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells may also contain genetic information in circular loops called episomes. Bacterial cells are very small, about the size of animal mitochondria (about 1-2 μm in diameter and 10 μm in length). Nucleated cells are characterized by three main shapes: rod-shaped, spherical and spiral. Instead of going through an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include Bacillus bacteria, E. coli bacteria　bacterium) and Salmonella bacteria ( Salmonella bacterium), but are not limited thereto.

The term "encode" as applied to a nucleic acid sequence refers to a polynucleotide that "encodes" a polypeptide in its native state or when manipulated by methods well known to those of ordinary skill in the art and is transcribed and/or translated. mRNA for the polypeptide and/or fragments thereof can be produced. The antisense strand is the complement of these nucleic acids, from which the coding sequence can be deduced.

The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological or cellular material, which is intended to provide a minimum of the desired structure or functionality (e.g., having a similar function or activity) while still retaining the desired structure or functionality. It is intended to have homology. When referring to an equivalent or biological equivalent to a reference polypeptide, protein or polynucleotide, it should be made clear that the equivalent or biological equivalent has the stated structural relationship with respect to the reference polypeptide, protein or polynucleotide and the equivalent or substantially equivalent biological activity. It should be understood that not mentioned. For example, non-limiting examples of equivalent polypeptides, proteins or polynucleotides are at least 60%, or alternatively at least 65% relative to or relative to a polypeptide, polynucleotide or protein sequence over the length of the reference polypeptide, polynucleotide or protein. , or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity Polypeptides, proteins or polynucleotides with Alternatively, an equivalent polypeptide is one that hybridizes under conditions of high stringency to a polynucleotide encoding such a reference polypeptide sequence and is encoded by a polynucleotide that has substantially equivalent or equivalent biological activity, or its complement. High stringency conditions are described herein and incorporated herein by reference. Alternatively, the equivalent thereof is at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively, over the length of the reference polynucleotide relative to the reference polynucleotide, e.g., a wild-type polynucleotide. A polypeptide encoded by a polynucleotide or its complement having at least 85%, or alternatively at least 90%, or alternatively at least 95% identity, or at least 97% sequence identity. Such equivalent polypeptides have the same biological activity as the polypeptide encoded by the reference polynucleotide.

Non-limiting examples of equivalent polynucleotides are at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, relative to the reference polynucleotide. or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 97% identity. Equivalent is also intended a polynucleotide or its complement that hybridizes under conditions of high stringency to a reference polynucleotide. Such equivalent polynucleotides have the same biological activity as the reference polynucleotide.

Polynucleotides or polynucleotide regions (or polypeptides or polypeptide regions) that have a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence, when aligned, refer to It means that the percentage of bases (or amino acids) is the same when comparing two sequences over the length of a polynucleotide. Alignment and percent homology or sequence identity can be performed using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds 1987) Supplement 30, section 7.7.18, Table 7.7.1. can be determined using A non-limiting exemplary alignment program is BLAST using default parameters. In particular, exemplary programs include BLASTN and BLASTP using the following default parameters: genetic code=standard; filter=none; strand=nboth; cutoff=60; expected=10; matrix=BLOSUM62; Description = 50 sequences; classification=high score; Database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbimnihgov/cgi-bin/BLAST. Sequence identity and percent identity were determined by incorporating them into clustalW (web address: genomejp/tools/clustalw/, last accessed on January 13, 2017) or Clustal Omega (available at ebiacuk/Tools/msa/clustalo/). can be determined

“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

As used herein, the term "at least 90% identical" refers to about 90% to about 100% identity of two compared sequences (polynucleotides or polypeptides). It is also at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, about 91% to about 100%, about 92% to about 92% About 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100 %, or from about 99% to about 100% identity.

As used herein, the terms "comprises," "is similar to," and "similar to" are used interchangeably while describing a function, activity, or functional activity of a polynucleotide, protein, and/or peptide, and refer to a protein, polynucleotide, and/or peptide. / or at least about 20% (at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%) of the activity of the peptide , at least about 97%, or about 100%) functional activity.

“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein bonding, or in any other sequence-specific manner. The complex may include two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a broader process, such as initiation of a PCR reaction or enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include an incubation temperature of about 25° C. to about 37° C.; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a washing solution from about 4xSSC to about 8xSSC. Examples of suitable hybridization conditions include an incubation temperature of about 40° C. to about 50° C.; a hybridization buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and about 5xSSC to about 2xSSC wash solution. Examples of high stringency conditions include an incubation temperature of about 55° C. to about 68° C.; a hybridization buffer concentration of about 1 x SSC to about 0.1 x SSC; a formamide concentration of about 55% to about 75%; and a washing solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, the hybridization incubation time is from 5 minutes to 24 hours with one, two or more wash steps, and the wash incubation time is about 1, 2 or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It should be understood that equivalents of SSC using other buffer systems may be used. In one aspect, an equivalent polynucleotide is one that hybridizes under stringent conditions to a reference polynucleotide or its complement. In another aspect, an equivalent polypeptide is a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a reference polynucleotide or its complement.

As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed into mRNA and/or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. When the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

As used herein, the term "functional" can be used to modify any molecular, biological, or cellular material with the intention of achieving a particular particular effect.

As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Accordingly, the term refers to single-, double-, or multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, or purine and pyrimidine bases or other natural, chemically or biochemically modified , polymers comprising non-natural, or derivatized nucleotide bases. In certain embodiments, a polynucleotide comprises and/or encodes messenger RNA (mRNA), short hairpin RNA, and/or small hairpin RNA. In one embodiment, the polynucleotide is or encodes an mRNA. In certain embodiments, the polynucleotide is double-stranded (ds) DNA, eg engineered ds DNA or ds cDNA synthesized from single-stranded RNA.

The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense refer to compounds of two or more subunits of amino acids, amino acid analogs or peptidomimetics. Subunits may be linked by peptide bonds. In another aspect, the subunits may be linked by other linkages, such as esters, ethers, and the like. A protein or peptide must contain at least 2 amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. As used herein, the term “amino acid” refers to natural and/or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs and peptidomimetics.

A contiguous amino acid sequence as used herein refers to a sequence having at least two amino acids. Note, however, that the contiguous amino acid sequence of the first part and the second part does not limit the amino acid sequence such that the first part is directly conjugated to the second part. It is also possible for a first part to be connected to a third part, eg to a second part via a link, thus forming one contiguous amino acid sequence.

As used herein, the terms “conjugate,” “conjugated,” “conjugating,” and “conjugation” refer to the formation of a linkage between molecules, particularly between two amino acid sequences and/or two polypeptides. Conjugation can be direct (ie, binding) or indirect (ie, through an additional molecule). Conjugation can be covalent or non-covalent.

As used herein, a contiguous amino acid sequence may include two or more polypeptides joined directly or indirectly (eg, via a linker) to each other.

As used herein, the term "recombinant expression system" refers to a genetic construct(s) for the expression of specific genetic material formed by recombination.

A “gene transfer vehicle” is defined as any molecule capable of delivering an inserted polynucleotide into a host cell. Examples of gene delivery vehicles include micelles, which are biocompatible polymers including liposomes, natural and synthetic polymers; lipoprotein; lipid nanoparticles; polypeptide; polysaccharides; lipopolysaccharide; artificial viral envelope; metal particles; and bacteria, or viruses such as rabies viruses, flaviviruses, lentiviruses, baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and expression in a variety of eukaryotic and prokaryotic hosts. It is another recombinant vehicle typically used in the art and can be used for simple protein expression as well as gene therapy.

The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, “gene delivery,” “gene transfer,” “mRNA-based delivery,” “transducing,” and the like refer to any method used for introduction into a host cell. A term that refers to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) of Such methods may be accomplished using a variety of well-known techniques, such as vector-mediated gene delivery (eg viral infection/transfection, or eg protamine complexes, lipid nanoparticles, polymer nanoparticles, lipid-polymer hybridization). nanoparticles, and inorganic nanoparticles, or a variety of other protein-based or lipid-based gene delivery complexes including combinations thereof) as well as technologies that facilitate the delivery of "naked" polynucleotides (eg eg electroporation, "gene gun" delivery, and various other techniques used for introduction of polynucleotides). An introduced polynucleotide may be unmodified or may contain one or more modifications; For example, modified mRNAs can be ARCA capped; enzymatic polyadenylation to add a tail of 100-250 adenosine residues (SEQ ID NO: 22); and substitution of one or both of the cytidines with 5-methylcytidine and/or uridine with pseudouridine. An introduced polynucleotide may be stably or transiently maintained in a host cell. Stable maintenance typically requires that the introduced polynucleotide contains an origin of replication compatible with the host cell or a replicon of the host cell, such as an extrachromosomal replicon (eg a plasmid) or a nuclear or mitochondrial replicon. It requires integration into a chromosome. A number of vectors are known in the art and capable of mediating the transfer of genes into mammalian cells as described herein.

A "plasmid" is an extrachromosomal DNA molecule isolated from chromosomal DNA that is capable of replicating independently of chromosomal DNA. In many cases, they are circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically provide a selective advantage under given environmental conditions. Plasmids can carry genes conferring resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the resulting protein can act as a toxin under similar circumstances.

A "plasmid" used for genetic engineering is referred to as a "plasmid vector". Many plasmids are commercially available for this use. The gene to be cloned is inserted into a copy of a plasmid containing the gene that renders the cell resistant to certain antibiotics and multiple cloning sites (MCS, or polylinkers), which allows for the facile insertion of DNA fragments at these locations. is a short region containing restriction sites used as Another major use of plasmids is to make large amounts of protein. In this case, researchers grow bacteria containing a plasmid carrying the gene of interest. Just as bacteria produce proteins that confer their antibiotic resistance, they can also be induced to produce large amounts of proteins from inserted genes.

"Yeast artificial chromosome" or "YAC" refers to vectors used for cloning large DNA fragments (greater than 100 kb and less than 3000 kb). It is an artificially constructed chromosome and contains telomeric, centromeric, and origin of replication sequences necessary for replication and preservation in yeast cells. Building using an initial circular plasmid is linearized using restriction enzymes, then DNA ligase can add the sequence or gene of interest into the linear molecule by use of cohesive ends. Yeast expression vectors such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids) are very useful because yeast is itself a eukaryotic cell, allowing eukaryotic protein products with post-translational modifications to be obtained, YAC was found to be more labile than BAC, producing a chimeric effect.

As used herein, the term “viral capsid” or “capsid” refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term “encapsidated” means enclosed within a viral capsid.

As used herein, the term "helper" in reference to a virus or plasmid is used to provide additional components necessary for replication and packaging of a viral particle or recombinant viral particle, e.g., a modified AAV disclosed herein. Refers to a virus or plasmid. Components encoded by helper viruses may include any genes required for virion assembly, encapsidation, genome replication, and/or packaging. For example, helper viruses may encode enzymes necessary for replication of the viral genome. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

A biological sample, or sample, as used herein may be obtained from a subject, cell line, or cultured cell or tissue. Exemplary samples include cell samples, tissue samples, liquid samples such as blood and other liquid samples of biological origin (ocular fluids (aqueous and vitreous humor), peripheral blood, serum, plasma, ascites, Urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous fluid, amniotic fluid, cerebrospinal fluid, breast milk, broncheoalveolar lavage fluid, semen, prostate fluid, cow's fluid or pre-ejaculatory fluid, female ejaculatory fluid, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, lime, chyle, bile, interstitial fluid, menstruation, pus, sebum, include but are not limited to vomiting, vaginal discharge/flushing, synovial fluid, mucosal secretions, feces, pancreatic juice, lavage fluid from the sinus cavity, bronchopulmonary aspirate, blastosyl cavity fluid, or umbilical cord blood.

As used herein, the term “detectable marker” refers to at least one marker capable of directly or indirectly producing a detectable signal. A non-exhaustive list of such markers includes, for example, colorimetric, fluorescence, luminescence, e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose 6 phosphate dehydrogenase, chromophore, It includes, for example, an enzyme that produces a signal detectable by fluorescence, luminescent dyes, electron microscopy or by its electrical properties, such as conductivity, amperometry, voltammetry, impedance, e.g. a molecule thereof A detectable group having a size sufficient to induce a detectable modification in physical and/or chemical properties is selected from optical methods such as diffraction, surface plasmon resonance, surface fluctuations, contact angle changes or physical methods such as atomic force spectroscopy, tunnel effect. , or by radioactive molecules such as 32P, 35S, 89Zr or ¹²⁵ I.

As used herein, the term “purification marker” refers to at least one marker useful for purification or identification. A non-exhaustive list of these markers is His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap , HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, Cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red (Texas Red), rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.

As used herein, an epitope tag is a biological structure or sequence that serves as an antigen recognized by an antibody, such as a protein or carbohydrate. In certain embodiments, epitope tags are used interchangeably with purification markers and/or affinity tags.

A "composition" is a combination of two or more compounds, e.g., active polypeptides, polynucleotides, viral vectors or antibodies, and another compound or composition, inactive (e.g., detectable label) or active (e.g., gene transfer vehicle). ) is intended to mean a combination of

"Pharmaceutical composition" is intended to include a combination of an active polypeptide, polynucleotide or antibody with a carrier, inactive or active, eg a solid support, which is intended to include the composition for diagnostic or therapeutic use in vitro, in vivo or ex vivo. make it fit

As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions such as oil/water or water/oil emulsions, and various Covers types of humectants. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm Sci, 15th Ed (Mack Publ Co, Easton).

"Subject", "individual" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, non-human primates, murines, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, and primates, particularly humans. don't In addition to being useful for human treatment, the present invention is also useful for veterinary treatment of companion mammals, exotic animals and livestock, including mammals, rodents, and the like. In one embodiment, mammals include horses, dogs and cats. In another embodiment of the invention, the human is an adolescent or toddler under the age of 18 years.

"Treatment" or "treatment" of a disease means (1) preventing a disease in a patient who may be predisposed to the disease but who is not yet experiencing or displaying symptoms of the disease, i.e., preventing clinical symptoms of the disease from occurring. ; (2) inhibiting the disease, ie arresting or reducing the occurrence of the disease or its clinical symptoms; or (3) alleviating the disease, ie causing regression of the disease or its clinical symptoms. In one aspect, the term "treatment" excludes prevention or prophylaxis.

The term "suffering" in relation to the term "treatment" refers to a patient or individual who has been diagnosed as suffering from or susceptible to a disease.

An "effective amount" is an amount sufficient to achieve beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery depends on a number of variables, including the length of time the individual dosage unit is used, the bioavailability of the therapeutic agent, the route of administration, and the like. However, the specific dosage level of a therapeutic agent of the present invention for any particular subject depends on the activity of the particular compound employed, the age, weight, general health, sex, and diet of the subject, the time of administration, rate of excretion, drug combination, and the combination of drugs being treated. It should be understood that this will depend on a variety of factors including the severity of the particular disorder and the form of administration. Therapeutic dosages can generally be titrated to optimize safety and efficacy. In one aspect, an effective amount is a therapeutically effective amount. Typically, dose-effect relationships from in vitro and/or in vivo studies can initially provide useful guidance on appropriate dosages for patient administration. Generally, one will attempt to administer an amount of compound effective to achieve serum levels commensurate with the concentration found to be effective in vitro. Determination of these parameters is within the ordinary skill in the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks. Consistent with this definition as used herein, the term "therapeutically effective amount" is an amount sufficient to inhibit RNA viral replication ex vivo, in vitro or in vivo.

The term administration includes, but is not limited to oral, parenteral (eg intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasal, vaginal, rectal, sublingual, urethral ( eg urethral suppositories) or by topical routes of administration (eg gels, ointments, creams, aerosols, etc.), alone or in combination, conventional non-toxic (non-toxic) appropriate for each route of administration. toxic) in suitable dosage unit formulations containing pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles. The present invention is not limited by route of administration, formulation or schedule of administration.

As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells whose specific function is served by co-infecting a cofactor virus. Currently, there are 13 serotypes of AAV that have been characterized. General information and reviews of AAV can be found, for example, in Carter, Handbook of Parvoviruses 1:169-228, 1989 and Berns, Virology 1743-1764, 1999. However, these same principles are fully expected to be applicable to additional AAV serotypes as it is well known that the various serotypes are structurally and functionally very closely related at the genetic level. See, eg, Blacklowe, Parvoviruses and Human Disease 165-174, 1988, J R Pattison, ed; and "Rose, Comprehensive Virology 3:1-61, 1974". For example, all AAV serotypes display very similar replication properties mediated by apparently homologous rep genes; All have three related capsid proteins as expressed in AAV2. The degree of relatedness can be assessed by heteroduplex analysis indicating extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

As used herein, the term "AAV expression cassette" refers to a nucleotide sequence comprising one or more polynucleotides (or transgenes) of interest flanked by AAV terminal repeat sequences (ITRs). Such AAV expression cassettes can be replicated and packaged into infectious viral particles (eg AAV vectors) when present in host cells transfected with vectors encoding and expressing the rep and cap gene products.

"AAV virion" or "AAV vector" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV expression cassette. When a particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply "AAV vector". Thus, production of an AAV vector particle necessarily includes production of an AAV expression cassette, since such plasmid is contained within the AAV vector particle.

Adeno-associated virus (AAV) is a replication-defective parvovirus, whose single-stranded DNA genome is approximately 4.7 kb in length, including a 145 nucleotide inverted terminal repeat (ITR). Multiple serotypes of AAV exist. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is described in Srivastava et al., J Virol, 45:555 as corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). -564 (1983)”. As another example, the complete genome of AAV-1 is provided under GenBank accession number NC_002077; The complete genome of AAV-3 is provided under Genbank Accession No. NC_1829; The complete genome of AAV-4 is provided under Genbank Accession No. NC_001829; The complete genome of AAV-5 is provided under Genbank Accession No. AF085716; The complete genome of AAV-6 is provided under Genbank Accession No. NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively (see also US Pat. Nos. 7,282,199 and 7,790,449 for AAV-8); The AAV-9 genome is provided by Gao et al., J Virol, 78:6381-6388 (2004); The AAV-10 genome is provided in Mol Ther, 13(1):67-76 (2006); The AAV-11 genome is provided in Virology, 330(2):375-383 (2004). Cloning of the AAVrh74 serotype is described in Rodino-Klapac, et al., Journal of translational medicine 5, 45 (2007). Contained within the ITRs are cis-acting sequences that direct viral DNA replication (rep), encapsidation/packaging and host cell chromosomal integration. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) coupled with differential splicing of a single AAV intron (e.g. at AAV2 nucleotides 2107 and 2227) generate four rep proteins (rep 78, rep 68, rep 52, and rep 40). The Rep protein possesses multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

A recombinant AAV genome of the present disclosure comprises a nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. AAV DNA within the rAAV genome is composed of AAV serotypes AAVrh74, AAVrh10, AAVrh20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9 , AAV-10, AAV-11, AAV-12 and AAV-13, including but not limited to, from any AAV serotype from which recombinant viruses can be derived. Production of pseudotyped rAAV is disclosed, for example, in WO 2001/83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). As noted in the background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. In some embodiments, to promote skeletal muscle specific expression, AAV1, AAV6, AAV8 or AAVrh74 is used.

As used in this specification and claims, the singular forms “a, an” and “the” refer to plural unless the context clearly dictates otherwise. For example, the term "cell" means a plurality of cells, including mixtures thereof.

As used herein, the terms "comprising" or "comprising" should be understood to mean that the compositions and methods include the recited elements but do not exclude others. “Consisting essentially of” when used to define compositions and methods means excluding any other elements of essential importance to the combination for the stated purpose. Thus, compositions consisting essentially of the elements as defined herein do not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like. "Isolating" shall mean excluding more than trace elements of other components and substantial process steps for administering a composition of the present invention to produce the composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

The term "isolated" as used herein in reference to a nucleic acid, eg DNA or RNA, refers to a molecule that has been separated from other DNA or RNA, respectively, present in the macromolecule's natural source. The term "isolated nucleic acid" is meant to include nucleic acid fragments that do not occur naturally as fragments. The term "isolated" is also used herein to refer to polypeptides, proteins and/or host cells, encompassing both polypeptides and recombinant polypeptides that have been isolated and purified from other cellular proteins. In another embodiment, the term "isolated" means separated from components, cells and otherwise normally associated cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies or fragment(s) thereof in nature. For example, an isolated cell is a cell that has been separated from a tissue or cell of a dissimilar phenotype or genotype. As will be apparent to those skilled in the art, non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies or fragment(s) thereof do not require "isolation" to distinguish them from their naturally occurring counterparts. .

The term “recombinant” as used herein in reference to a polypeptide or polynucleotide, such as DNA or RNA, refers to a molecule formed by laboratory methods of recombination, such as molecular cloning. Molecular cloning techniques are known in the art and include PCR amplification of polynucleotides, enzymatic digestion of polynucleotides, ligation of polynucleotides into expression cassettes (eg mammalian expression cassettes), transformation, transfection or transduction of cells, and expression of polynucleotides to produce polypeptides, but is not limited thereto. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 2012. The term “recombinant polynucleotide” is meant to include fragments of protein-encoding polynucleotides. For example, the recombinant polynucleotide may include a fragment of a polynucleotide encoding the human dysferrin protein. Recombinant polynucleotides can be produced by PCR amplification of fragments of protein-encoding polynucleotides. Recombinant polypeptides can be produced by expression of one or more recombinant polynucleotides.

Polynucleotides encoding fragments of the human dysferrin (hDSYSF) protein are disclosed herein. Further disclosed herein are plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions comprising polynucleotides encoding fragments of the human dysferrin (hDSYSF) protein. Methods of making and using such polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions are also disclosed herein.

Disclosed herein is a recombinant polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises (a) SEQ ID NOs: 1, 6 , or a nucleotide sequence of 18; (b) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 1, 6, or 18 over the entire length of each of SEQ ID NO: 1, 6, or 18; 96%, 97%, 98%, or 99% identical nucleotide sequences; (c) the nucleotide sequence of SEQ ID NO: 13 or 15; (d) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; 98%, or 99% identical nucleotide sequences; (e) a nucleotide sequence encoding the hDYSF protein, wherein the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (f) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of (e) over the entire length of the nucleotide sequence of (e). %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences;

Further disclosed herein is a recombinant polynucleotide sequence encoding a fragment of the human dysferrin protein, wherein the recombinant polynucleotide comprises a first nucleotide sequence, wherein the first nucleotide sequence comprises (a) SEQ ID NOs: 2, 8 , or a nucleotide sequence of 19; (b) at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 2, 8, or 19 over the entire length of each of SEQ ID NO: 2, 8, or 19; 96%, 97%, 98%, or 99% identical nucleotide sequences; (c) the nucleotide sequence of SEQ ID NO: 14 or 16; (d) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences; (e) a polynucleotide sequence encoding a hDYSF protein, wherein the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (f) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of the polynucleotide sequence of (e) over the entire length of the nucleotide sequence of (e); 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences;

Further disclosed herein are adeno-associated virus (AAV) vectors. In some embodiments, an adeno-associated virus (AAV) vector comprises (a) a first inverted terminal repeat (ITR); (b) a polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the polynucleotide comprises (i) a nucleotide sequence of SEQ ID NO: 1 or 6; (ii) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6; a nucleotide sequence that is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; (iii) the nucleotide sequence of SEQ ID NO: 13 or 15; (iv) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15; a nucleotide sequence that is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; (v) a nucleotide sequence encoding hDYSF protein, wherein the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences;

In some embodiments, an adeno-associated virus (AAV) vector is

(a) a first inverted terminal repeat (ITR);

(b) a polynucleotide encoding a fragment of human dysferrin (hDYSF) protein, wherein the polynucleotide comprises (i) a nucleotide sequence of SEQ ID NO: 2 or 8; (ii) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8; a nucleotide sequence that is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; (iii) the nucleotide sequence of SEQ ID NO: 14 or 16; (iv) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16; a nucleotide sequence that is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; (v) a nucleotide sequence encoding a hDYSF protein, wherein the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences; and

(c) a second ITR;

Including, wherein the polynucleotide is flanked by the first ITR and the second ITR.

Further disclosed herein are adeno-associated virus (AAV) expression cassettes. In some embodiments, an AAV expression cassette comprises (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any of the ITRs disclosed herein; (b) any of the 5' hDYSF polynucleotides disclosed herein; and (c) a second ITR, wherein the second ITR comprises any of the ITRs disclosed herein; wherein the 5' hDYSF polynucleotide of (b) is one of (a) and (c) above It is flanked by a first ITR and a second ITR.

In some embodiments, an AAV expression cassette comprises (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any of the ITRs disclosed herein; (b) any of the 3' hDYSF polynucleotides disclosed herein; and (c) a second ITR, wherein the second ITR comprises any of the ITRs disclosed herein; wherein the 3' hDYSF polynucleotide of (b) is the second ITR of (a) and (c) It is flanked by 1 ITR and the 2nd ITR.

Further disclosed herein are adeno-associated virus (AAV) vectors. In some embodiments, an AAV vector comprises (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any ITR disclosed herein; (b) any 5' hDYSF polynucleotide disclosed herein; and (c) a second ITR, wherein the second ITR comprises any ITR disclosed herein; wherein the 5′ hDYSF polynucleotide of (b) is the first ITR of (a) and (c) above and flanked by a second ITR.

In some embodiments, an AAV vector comprises (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any ITR disclosed herein; (b) any 3' hDYSF polynucleotide disclosed herein; and (c) a second ITR, wherein the second ITR comprises any ITR disclosed herein; wherein the 3' hDYSF polynucleotide of (b) is the first ITR of (a) and (c) above and flanked by a second ITR.

Further disclosed herein is a dual adeno-associated virus (AAV) vector system. In some embodiments, the dual AAV vector system comprises (a) a first AAV vector, wherein the first AAV vector comprises any 5' hDYSF polynucleotide disclosed herein; and (b) a second AAV vector, wherein the second AAV vector comprises any 3' hDYSF polynucleotide disclosed herein.

Further disclosed herein is a dual adeno-associated virus (AAV) vector system. In some embodiments, the dual AAV vector system comprises (a) a first AAV vector, wherein the first AAV vector comprises, consists of, or consists essentially of any 5' hDYSF AAV vector disclosed herein; and (b) a second AAV vector, wherein the second AAV vector comprises, consists of, or consists essentially of any 3' hDYSF AAV vector disclosed herein; is essentially composed of

Further disclosed herein are adeno-associated virus (AAV) vectors. In some embodiments, an AAV vector comprises any 5' hDYSF polynucleotide disclosed herein.

In some embodiments, an AAV vector comprises any 3' hDYSF polynucleotide disclosed herein.

In some embodiments, polynucleotides, plasmids, viral vectors (eg viruses or viral particles), vector systems, viral packaging systems, cells, and compositions may include inverted terminal repeats (ITRs), promoters, introns, selectable markers , or one or more nucleotide sequences comprising, consisting of, or consisting essentially of an origin of replication (ORI).

In some embodiments, polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions comprise inverted terminal repeats (ITRs), selectable markers, origins of replication (ORIs), untranslated regions (UTRs) , or one or more additional nucleotide sequences comprising a polyadenylation (polyA) signal.

Further disclosed herein are methods of treating dysperinopathies. In some embodiments, the method of treating dysferinopathy comprises administering to a subject in need thereof any of the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions disclosed herein. do.

Further disclosed herein is the use of any of the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions disclosed herein in the manufacture of a medicament for the treatment of dysferinopathy.

Homologous recombination and hDYSF snippet

AAV-mediated gene therapy represents a desirable treatment strategy for a number of diseases, but it is hampered by the restrictive 4.7 kb packaging limitation of AAV virions. Of particular interest are diseases that are currently incurable or do not have an effective therapy, such as dysperinopathies. Methods for making or producing a full-length dysferrin gene by homologous recombination of two partial genomes are disclosed herein. In one example, two partially packaged genomes are shown in Figure 1 as pAAV.MHCK7.DYSF5'.PTG and pAAV.DYSF3'.POLYA. When the two genomes are packaged into an AAV vector and delivered to a cell (eg myocyte), whether via viral transfer or via a non-viral method (eg LNP), they contain the full-length dysferrin coding region. By generating a transcript that contains, expression of a functional dysferrin protein is induced. The overlapping region between the two polynucleotides facilitates homologous recombination leading to a transcript containing the full-length dysferrin gene. By separating the full-length dysferrin gene into two partially packaged genomes, the method successfully bypasses AAV packaging restrictions and produces a functional full-length dysferrin gene. In one embodiment, the transcript comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 20. It is an expression cassette that In one embodiment, the transcript is an expression cassette comprising the sequence of SEQ ID NO:20.

In some embodiments, disclosed herein is a recombinant polynucleotide encoding human dysferrin (hDYSF) protein, wherein the recombinant polynucleotide sequence is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences. In some cases, recombinant polynucleotides comprising the nucleotide sequence of SEQ ID NO: 20 are disclosed herein. In some instances, methods of making recombinant polypeptides are disclosed herein. In some cases, the method includes contacting the cell with a recombinant polynucleotide encoding a 5' fragment of hDYSF protein and a second recombinant polynucleotide encoding a 3' fragment of hDYSF protein. In some cases, the methods include contacting the cells with a dual AAV vector system described herein. In some cases, the cells are eukaryotic cells, optionally muscle cells, cardiac cells, and/or liver cells. In some cases, the method comprises administering to the subject a recombinant polynucleotide encoding a 5' fragment of hDYSF protein and a second recombinant polynucleotide encoding a 3' fragment of hDYSF protein. In some cases, the methods include administering to the subject a dual AAV vector system described herein. In some cases, the method is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or the nucleotide sequence of SEQ ID NO: 20. treating muscular dystrophy in a subject by expressing a recombinant polynucleotide comprising the nucleotide sequence. In some cases, the subject has a dysferinopathy optionally selected from LGMD2B or Miyoshi myopathy.

Also disclosed herein are two recombinant polynucleotides, each encoding a fragment of the human dysferrin (hDYSF) protein, capable of directing production of the full-length dysferrin gene by homologous recombination as described above. In some embodiments, the recombinant polynucleotide encodes a 5' fragment of the hDYSF protein. In some embodiments, a recombinant polynucleotide encoding a 5' fragment of an hDYSF protein is referred to as a 5' hDYSF polynucleotide. In some embodiments, the recombinant polynucleotide encodes a 3' fragment of the hDYSF protein. In some embodiments, a recombinant polynucleotide encoding a 3' fragment of hDYSF protein is referred to as a 3' hDSYF polynucleotide.

5' hDYSF polynucleotide

In some embodiments, a 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150 of SEQ ID NO: 11 -3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000 ; -3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 3330-3365, 3330-3360, 3330-3355, 3335-3365, 3335- It comprises, consists of, or consists essentially of between 3350, 3340-3365, 3340-3360, or 3340-3355 contiguous nucleotides. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 3360, 3359, 3358, 3357, 3356, 3355, 3354, 3353, 33512, 33512, 377-3716 , 3350, 3349, 3348, 3347, 3346, 3345, 3344, 3343, 3342, 3341, or 3340 or less consecutive nucleotides, consisting of, or consisting essentially of. . In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 3330-3365, 3330-3360, 3330-3355, 3335-3365, 3335-3350 in the range between 377-3716 , 3340-3365, 3340-3360, or 3340-3355 contiguous nucleotides, wherein the 5' hDYSF polynucleotide comprises nucleotides 100-4000 of SEQ ID NO: 11 ,100-3900,100-3800,100-3750,100-3716,150-4000,150-3900,150-3800,150-3750,150-3716,200-4000,200-3900,200-3800,200 -3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000 , 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370-3750, 370-3716, 377-4000, 377 at least 80%, 82%, 85%, 87%, 88%, 90% of the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between -3900, 377-3800, 377-3750, or 377-3716; 92%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 3360, 3359, 3358, 3357, 3356, 3355, 3354, 3353, 33512, 33512, 377-3716 , 3350, 3349, 3348, 3347, 3346, 3345, 3344, 3343, 3342, 3341, or 3340 or less consecutive nucleotides, consisting of, or consisting essentially of, wherein said 5' The hDYSF polynucleotide has nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150-3750, 150-3716, 200 of SEQ ID NO: 11 -4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800 , 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, nucleotide sequence of SEQ ID NO: 11 and at least 80 %, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or at least 85% relative to the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between 377-3716. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or at least 90% relative to the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between 377-3716. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or at least 95% relative to the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between 377-3716. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 3330-3365, 3330-3360, 3330-3355, 3335-3365, 3335-3350 in the range between 377-3716 , 3340-3365, 3340-3360, or 3340-3355 consecutive nucleotide mismatches between, consisting of, or consisting essentially of, wherein the 5' hDYSF polynucleotide is SEQ ID NO: 11 nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150-3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300- 3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370-3750, 370-37 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 in the range between 16, 377-4000, 377-3900, 377-3800, 377-3750, or 377-3716 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less consecutive nucleotide mismatches. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 3360, 3359, 3358, 3357, 3356, 3355, 3354, 3353, 33512, 33512, 377-3716 , 3350, 3349, 3348, 3347, 3346, 3345, 3344, 3343, 3342, 3341, or 3340 or less consecutive nucleotides, consisting of, or consisting essentially of, wherein said 5' The hDYSF polynucleotide has nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150-3750, 150-3716, 200 of SEQ ID NO: 11 -4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800 , 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 30, 25, 20, 19, 18, 17, 16 in the range between 370-3800, 370-3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 377-3716 , 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less consecutive nucleotides, consisting essentially of these consists of In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 15 or fewer nucleotide mismatches in the region between 377-3716. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 10 or fewer nucleotide mismatches in the region between 377-3716. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 5 or fewer nucleotide mismatches in the region between 377-3716. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- contains 1 nucleotide mismatch in the region between 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or 377-3716. In some embodiments, the 5' hDYSF polynucleotide comprises nucleotides 100-4000, 100-3900, 100-3800, 100-3750, 100-3716, 150-4000, 150-3900, 150-3800, 150- 3750, 150-3716, 200-4000, 200-3900, 200-3800, 200-3750, 200-3716, 250-4000, 250-3900, 250-3800, 250-3750, 250-3716, 300-4000, 300-3900, 300-3800, 300-3750, 300-3716, 350-4000, 350-3900, 350-3800, 350-3750, 350-3716, 370-4000, 370-3900, 370-3800, 370- 3750, 370-3716, 377-4000, 377-3900, 377-3800, 377-3750, or at least one nucleotide mismatch in the region between 377-3716. In some embodiments, the 5' polynucleotide comprises at least one nucleotide mismatch in the region between nucleotides 377-3716 of SEQ ID NO: 11. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide is 3330-3365, 3330-3360, 3330-3355, 3335-3365, 3335-3350, 3340-3365, 3340-3360, or 3340-3355 contiguous nucleotides of SEQ ID NO: 11 comprising, consisting of, or consisting essentially of, wherein the 5' hDYSF polynucleotide is at nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550 of SEQ ID NO: 11 ; -3700, 3371-3650, 3371-3600, 3371-3550, or the region containing 3371-3500. In some embodiments, the 5' hDYSF polynucleotide is 3360, 3359, 3358, 3357, 3356, 3355, 3354, 3353, 3352, 3351, 3350, 3349, 3348, 3347, 3346, 3345, 33434, 33434 of SEQ ID NO: 11 , 3342, 3341, or 3340 or fewer contiguous nucleotides, consisting of, or consisting essentially of, wherein the 5' hDYSF polynucleotide is at nucleotide positions 3400-3716, 3400 of SEQ ID NO: 11 -3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390-3650, 3390-3600, 3390-3550, 3390-3500, 3380-303716 , 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-3600, 3371-3550, or 3371-3500. In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or at least 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% of the nucleotide sequence of the region over the entire length of the region comprising 3371-3500 , or 100% identical nucleotide sequences. In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of the region over the entire length of the region comprising 3371-3500. In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or a nucleotide sequence that is at least 95% identical to the nucleotide sequence of the region over the entire length of the region comprising 3371-3500. In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or a nucleotide sequence that is at least 99% identical to the nucleotide sequence of the region over the entire length of the region comprising 3371-3500. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less nucleotide mismatches to the nucleotide sequence of the region comprising 3371-3500. In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or 5 or fewer nucleotide mismatches to the nucleotide sequence of the region comprising 3371-3500. In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or 2 or fewer nucleotide mismatches to the nucleotide sequence of the region comprising 3371-3500. In some embodiments, the 5' hDYSF polynucleotide has nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390 of SEQ ID NO: 11 -3650, 3390-3600, 3390-3550, 3390-3500, 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-350710 , or 1 or less nucleotide mismatches to the nucleotide sequence of the region comprising 3371-3500. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide is 3360, 3359, 3358, 3357, 3356, 3355, 3354, 3353, 3352, 3351, 3350, 3349, 3348, 3347, 3346, 3345, 33434, 33434 of SEQ ID NO: 11 , 3342, 3341, or 3340 or less contiguous nucleotides, wherein the 5' hDYSF polynucleotide comprises SEQ ID NO: 11 at nucleotide positions 3400-3716, 3400- 3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390-3650, 3390-3600, 3390-3550, 3390-3500, 3380-303716 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, 3371-3600, 3371-3550, or 3371-3500, wherein the 5' hDYSF polynucleotide is SEQ ID NO: 11 nucleotide positions 3400-3716, 3400-3700, 3400-3650, 3400-3600, 3400-3550, 3400-3500, 3390-3716, 3390-3700, 3390-3650, 3390-3600, 3390-3550, , 3380-3716, 3380-3700, 3380-3650, 3380-3500, 3371-3716, 3371-3700, 3371-3650, over the entire length of the region containing at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide is 3330-3365, 3330-3360, 3330-3355, 3335-3365, 3335-3350, 3340-3365, 3340-3360, or 3340-3355 contiguous nucleotides of SEQ ID NO: 11 comprising, consisting of, or consisting essentially of, wherein the 5' hDYSF polynucleotide comprises nucleotide positions 1-100, 1-200, 1-300, 1-350, 1-375 of SEQ ID NO: 11; 1-376, 100-200, 100-300, 100-350, 100-375, 100-376, 200-300, 200-350, 200-375, or the region containing 200-376 is not included. In some embodiments, the 5' hDYSF polynucleotide does not include a region comprising nucleotide positions 1-376 of SEQ ID NO: 11. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide is 3360, 3359, 3358, 3357, 3356, 3355, 3354, 3353, 3352, 3351, 3350, 3349, 3348, 3347, 3346, 3345, 33434, 33434 of SEQ ID NO: 11 , 3342, 3341, or 3340 contiguous nucleotides, wherein the 5' hDYSF polynucleotide comprises nucleotide positions 1-100, 1-200, 1- 300, 1-350, 1-375, 100-200, 100-300, 100-350, 100-375, 200-300, 200-350, 200-375. In some embodiments, the 5' hDYSF polynucleotide does not comprise a region consisting of nucleotide positions 1-376 of SEQ ID NO:11. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the 5' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, 89%, 90%, 91%, 92% of the nucleotide sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, consisting of, or consisting essentially of. In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1 over its entire length. In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 92% identical to the nucleotide sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1 over its entire length. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the 5' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, 89%, 90%, 91%, 92% of the nucleotide sequence of SEQ ID NO: 13 over the entire length of SEQ ID NO: 13 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, consisting of, or consisting essentially of. In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 13 over the entire length of SEQ ID NO: 13. In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 92% identical to the nucleotide sequence of SEQ ID NO: 13 over the entire length of SEQ ID NO: 13. In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 13 over the entire length of SEQ ID NO: 13. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, a 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding a fragment of hDYSF protein comprising the N-terminal region of wild-type hDSYF protein. In some embodiments, fragments of hDYSF protein comprising the N-terminal region of wild-type hDSYF protein are referred to as N-terminal hDYSF proteins. In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding an N-terminal hDYSF protein, wherein the N-terminal hDYSF protein comprises amino acids of SEQ ID NO: 12 Residues 1-1113, 200-1113, 400-1113, 500-1113, 600-1113, 650-113, 650-1100, 700-1100, 700-1113, 700-1050, 700-1000, 800-1113, 800 -1100, 800-1050, 900-1113, 900-1100, 1000-1113, or 1000-1100. In some embodiments, a 5' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, comprises, consists of, or consists essentially of 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; Wherein the N-terminal hDYSF protein comprises amino acid residues 1-1113, 200-1113, 400-1113, 500-1113, 600-1113, 650-113, 650-1100, 700-1100, 700- comprising, consisting of, or consisting essentially of 1113, 700-1050, 700-1000, 800-1113, 800-1100, 800-1050, 900-1113, 900-1100, 1000-1113, or 1000-1100; includes the area composed of them. In some embodiments, the N-terminal hDYSF protein comprises a region comprising, consisting of, or consisting essentially of amino acid residues 999-1113, 999-1100, 1000-1113, or 1000-1100. . In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding an N-terminal hDYSF protein, wherein the N-terminal hDYSF protein is at least 1000 amino acids in length wherein the N-terminal hDYSF protein comprises a region comprising amino acid residues 999-1113, 999-1100, 1000-1113, or 1000-1100 of SEQ ID NO: 12. In some embodiments, a 5' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, comprises, consists of, or consists essentially of 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; wherein the N-terminal hDYSF protein is at least 1000 amino acids in length, wherein the N-terminal hDYSF protein comprises amino acid residues 999-1113, 999-1100, 1000-1113, or 1000-1100 of SEQ ID NO: 12 contains the area In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding an N-terminal hDYSF protein, wherein the N-terminal hDYSF protein comprises amino acids of SEQ ID NO: 9 It comprises, consists of, or consists essentially of a sequence. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, a 5' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, comprises, consists of, or consists essentially of a nucleotide sequence that is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; Wherein the N-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the 5' hDYSF polynucleotide comprises or consists of a nucleotide sequence that is at least 90% identical to a nucleotide sequence encoding an N-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the N-terminal hDYSF protein is, or consists essentially of, wherein the N-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the 5' hDYSF polynucleotide comprises or consists of a nucleotide sequence that is at least 92% identical to a nucleotide sequence encoding an N-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the N-terminal hDYSF protein is, or consists essentially of, wherein the N-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the 5' hDYSF polynucleotide comprises or consists of a nucleotide sequence that is at least 95% identical to a nucleotide sequence encoding an N-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the N-terminal hDYSF protein is, or consists essentially of, wherein the N-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the 5' hDYSF polynucleotide comprises or consists of a nucleotide sequence that is at least 97% identical to a nucleotide sequence encoding an N-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the N-terminal hDYSF protein is, or consists essentially of, wherein the N-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the N-terminal hDYSF protein is at least 80%, 82%, 85%, 88%, 89%, 90%, 91%, 92% of the amino acid sequence of SEQ ID NO: 9 over the entire length of SEQ ID NO: 9 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences. In some embodiments, the N-terminal hDYSF protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9. In some embodiments, the N-terminal hDYSF protein comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9. In some embodiments, the N-terminal hDYSF protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9. In some embodiments, the N-terminal hDYSF protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO:9 over the entire length of SEQ ID NO:9. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises the nucleotide sequence of SEQ ID NO:6. In some embodiments, the 5' hDYSF polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 6 over the entire length of SEQ ID NO: 6 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the 5' hDYSF polynucleotide comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6. In some embodiments, the 5' hDYSF polynucleotide comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 5' hDYSF polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the 5' hDYSF polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 6 over the entire length of SEQ ID NO: 15 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the 5' hDYSF polynucleotide comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 15 over the entire length of SEQ ID NO: 15. In some embodiments, the 5' hDYSF polynucleotide comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 15 over the entire length of SEQ ID NO: 15. In some embodiments, the 5' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

3' hDYSF polynucleotide

In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 3500-4100, 3500-4000, 3500-3900, 3500-3880, 3500-3870, 3600-4 in the range of 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619 , 3600-4000, 3600-3900, 3600-3880, 3600-3870, 3700-4100, 3700-4000, 3700-3900, 3700-3880, 3700-3870, 3800-4100, 3800-4000- or 38000- comprises, consists of, or consists essentially of consecutive nucleotides between In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 4100, 4000, 3900, 3880, or 3870 or less contiguous nucleotides of the region between, or consisting of consists of, or consists essentially of. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750-6750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 3500-4100, 3500-4000, 3500-3900, 3500-3880, 3500-3870, 10600-4 in the range of 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619 , 3600-4000, 3600-3900, 3600-3880, 3600-3870, 3700-4100, 3700-4000, 3700-3900, 3700-3880, 3700-3870, 3800-4100, 3800-4000-, or 3800-38000 wherein the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600 of SEQ ID NO: 11 -6725, 2600-6700, 2700-6850, 2700-6800, 2700-6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6700, 61620 , 2750-6850, 2750-6800, 2750-6780, 2750 -6750, 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-2754, 2754-27540 , 2754-6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or at least 80%, 82%, 85 relative to the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between %, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750-6750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 comprises, consists of, or consists of 4100, 4000, 3900, 3880, or 3870 contiguous nucleotides of the region between 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619, or Consisting essentially of these, wherein the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800 of SEQ ID NO: 11 ; -6750, 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-2754, 2754-27540 , 2754-6700, 2754-6680, 2754-6650, at least 80%, 82%, 85%, 87%, 88%, 90%, 92% of the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between 2754-6625, 2754-6620, or 2754-6619 , 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750-6750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or at least 85% relative to the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between 2754-6619. In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750-6750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or at least 90% relative to the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between 2754-6619. In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750-6750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or at least 95% relative to the nucleotide sequence of SEQ ID NO: 11 over the entire length of the region between 2754-6619. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750-6750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 3500-4100, 3500-4000, 3500-3900, 3500-3880, 3500-3870, 10600-4 in the range of 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619 , 3600-4000, 3600-3900, 3600-3880, 3600-3870, 3700-4100, 3700-4000, 3700-3900, 3700-3880, 3700-3870, 3800-4100, 3800-4000-, or 3800-38000 wherein the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600 of SEQ ID NO: 11 -6725, 2600-6700, 2700-6850, 2700-6800, 2700-6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6700, 61620 , 2750-6850, 2750-6800, 2750-6780, 2750 -6750, 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-2754, 2754-27540 , 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less consecutive nucleotide mismatches. In some embodiments, the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700- 6780, 2700-6750, 2700-6725, 2700-6700, 2700-6680, 2700-6650, 2700-6625, 2700-6620, 2700-6619, 2750-6850, 2750-6800, 2750-6750, 67-250,6780 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-27 2754 6700, 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 4100, 4000, 3900, 3880, or 3870 or less contiguous nucleotides of the region between, or consisting of Consisting of, or consisting essentially of, wherein the 3' hDYSF polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850 of SEQ ID NO: 11 ; -6780, 2750-6750, 2750-6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-66780 , 2754-6725, 2754-6700, 2754-6680, 27 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 in any range between 54-6650, 2754-6625, 2754-6620, or 2754-6619 , 7, 6, 5, 4, 3, 2, 1 or less consecutive nucleotide mismatches. In some embodiments, the 5' polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700-6780 of SEQ ID NO: 11 ; -6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-670725 , 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619 in the region between 15 or less nucleotide mismatches. In some embodiments, the 5' polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700-6780 of SEQ ID NO: 11 ; -6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-670725 , 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619 in the region between 10 or less nucleotide mismatches. In some embodiments, the 5' polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700-6780 of SEQ ID NO: 11 ; -6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-670725 , 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619 in the region between 5 or less nucleotide mismatches. In some embodiments, the 5' polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700-6780 of SEQ ID NO: 11 ; -6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-670725 , 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619. In some embodiments, the 5' polynucleotide comprises nucleotides 2600-6850, 2600-6800, 2600-6780, 2600-6750, 2600-6725, 2600-6700, 2700-6850, 2700-6800, 2700-6780 of SEQ ID NO: 11 ; -6725, 2750-6700, 2750-6680, 2750-6650, 2750-6625, 2750-6620, 2750-6619, 2754-6850, 2754-6800, 2754-6780, 2754-6750, 2754-670725 , 2754-6680, 2754-6650, 2754-6625, 2754-6620, or 2754-6619. In some embodiments, the 5' polynucleotide comprises at least one nucleotide mismatch in the region between nucleotides 2754-6619 of SEQ ID NO: 11. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide is 3500-4100, 3500-4000, 3500-3900, 3500-3880, 3500-3870, 3600-4100, 3600-4000, 3600-3900, 3600-3880 of SEQ ID NO: 11 , 3600-3870, 3700-4100, 3700-4000, 3700-3900, 3700-3880, 3700-3870, 3800-4100, 3800-4000, or 3800-3900 contiguous nucleotides, or , or consisting essentially of these, wherein the 5' hDSYF polynucleotide comprises nucleotide positions 6620-6914, 6620-6900, 6620-6800, 6620-6700, 6700-6914, 6700-6800, 6800-6914 of SEQ ID NO: 11; or the region consisting of 6800-6900. In some embodiments, the 3' hDYSF polynucleotide does not comprise a region consisting of nucleotide positions 6620-6914 of SEQ ID NO:11. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of 4100, 4000, 3900, 3880, or 3870 or fewer contiguous nucleotides of SEQ ID NO: 11, The 3' hDSYF polynucleotide comprises a region consisting of nucleotide positions 6620-6914, 6620-6900, 6620-6800, 6620-6700, 6700-6914, 6700-6800, 6800-6914, or 6800-6900 of SEQ ID NO: 11 I never do that. In some embodiments, the 3' hDYSF polynucleotide does not comprise a region consisting of nucleotide positions 6620-6914 of SEQ ID NO:11. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the 3' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, 89%, 90%, 91%, 92% of the nucleotide sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, consisting of, or consisting essentially of. In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:2 over its entire length. In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 92% identical to the nucleotide sequence of SEQ ID NO:2 over its entire length. In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:2 over its entire length. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the 3' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, 89%, 90%, 91%, 92% of the nucleotide sequence of SEQ ID NO: 14 over the entire length of SEQ ID NO: 14 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, consisting of, or consisting essentially of. In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 14 over the entire length of SEQ ID NO: 14. In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 92% identical to the nucleotide sequence of SEQ ID NO: 14 over the entire length of SEQ ID NO: 14. In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 14 over the entire length of SEQ ID NO: 14. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, a 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding a fragment of an hDYSF protein comprising a C-terminal region of a wild-type hDSYF protein. In some embodiments, a fragment of a hDYSF protein comprising the C-terminal region of a wild-type hDSYF protein is referred to as a C-terminal hDYSF protein. In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding a C-terminal hDYSF protein, wherein the C-terminal hDYSF protein comprises amino acids of SEQ ID NO: 12 comprises, consists of, or consists essentially of residues 750-2080, 750-2000, 750-1900, 775-2080, 775-2000, 775-1900, 794-2080, 794-2000, or 794-1900; It includes a region composed of them. In some embodiments, the 3' hDYSF polynucleotide is at least 80%, 82%, 85%, 88%, comprises, consists of, or consists essentially of 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences; wherein the C-terminal hDYSF protein comprises amino acid residues 750-2080, 750-2000, 750-1900, 775-2080, 775-2000, 775-1900, 794-2080, 794-2000, or 794 of SEQ ID NO: 12 -Includes a region that contains, consists of, or consists essentially of 1900. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding a C-terminal hDYSF protein, wherein the C-terminal hDYSF protein has a length of 1400, 1350 , 1325, 1300, 1290, or 1287 or less amino acids, wherein the C-terminal hDYSF protein comprises amino acid residues 750-2080, 750 of SEQ ID NO: 12 -2000, 750-1900, 775-2080, 775-2000, 775-1900, 794-2080, 794-2000, or 794-1900. In some embodiments, the 3' hDYSF polynucleotide is at least 80%, 82%, 85%, 88% of the nucleotide sequence encoding the C-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the C-terminal hDYSF protein. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, consisting of, or consisting essentially of wherein the C-terminal hDYSF protein is 1400, 1350, 1325, 1300, 1290, or 1287 amino acids or less in length, and the C-terminal hDYSF protein comprises amino acid residues 750-2080 of SEQ ID NO: 12; 750-2000, 750-1900, 775-2080, 775-2000, 775-1900, 794-2080, 794-2000, or 794-1900. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding a C-terminal hDYSF protein, wherein the C-terminal hDYSF protein has a length of 1400, 1350 , 1325, 1300, 1290, or 1287 or less amino acids comprising, consisting of, or consisting essentially of, wherein the C-terminal hDYSF protein comprises amino acid residues 678-793 of SEQ ID NO: 12; It does not include regions containing 678-750, 678-725, or 678-700. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence encoding a C-terminal hDYSF protein, wherein the C-terminal hDYSF protein comprises amino acids of SEQ ID NO: 10 It comprises, consists of, or consists essentially of a sequence. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide is at least 80%, 82%, 85%, 88% of the nucleotide sequence encoding the C-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the C-terminal hDYSF protein. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences, consisting of, or consisting essentially of Wherein the C-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the 3' hDYSF polynucleotide comprises or consists of a nucleotide sequence that is at least 90% identical to the nucleotide sequence encoding the C-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the C-terminal hDYSF protein. Consisting of, or consisting essentially of, wherein the C-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the 3' hDYSF polynucleotide comprises or consists of a nucleotide sequence that is at least 92% identical to the nucleotide sequence encoding the C-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the C-terminal hDYSF protein. Consisting of, or consisting essentially of, wherein the C-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the 3' hDYSF polynucleotide comprises or consists of a nucleotide sequence that is at least 95% identical to the nucleotide sequence encoding the C-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the C-terminal hDYSF protein. Consisting of, or consisting essentially of, wherein the C-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the 3' hDYSF polynucleotide comprises, or consists of, a nucleotide sequence that is at least 97% identical to a nucleotide sequence encoding a C-terminal hDYSF protein over the entire length of the nucleotide sequence encoding the C-terminal hDYSF protein. Consisting of, or consisting essentially of, wherein the C-terminal hDYSF protein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the C-terminal hDYSF protein is at least 80%, 82%, 85%, 88%, 89%, 90%, 91%, 92% of the amino acid sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences. In some embodiments, the C-terminal hDYSF protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10. In some embodiments, the C-terminal hDYSF protein comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10. In some embodiments, the C-terminal hDYSF protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10. In some embodiments, the C-terminal hDYSF protein comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 10 over the entire length of SEQ ID NO: 10. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises the nucleotide sequence of SEQ ID NO:8. In some embodiments, the 3' hDYSF polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 8 over the entire length of SEQ ID NO: 8 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the 3' hDYSF polynucleotide comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:8 over the entire length of SEQ ID NO:8. In some embodiments, the 3' hDYSF polynucleotide comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:8 over the entire length of SEQ ID NO:8. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the 3' hDYSF polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the 3' hDYSF polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 16 over the entire length of SEQ ID NO: 16 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the 3' hDYSF polynucleotide comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 16 over the entire length of SEQ ID NO: 16. In some embodiments, the 3' hDYSF polynucleotide comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 16 over the entire length of SEQ ID NO: 16. In some embodiments, the 3' hDYSF polynucleotide does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein.

In some embodiments, the sequences of the 5' hDSYF polynucleotide and the 3' hDSYF polynucleotide comprise an overlap of at least 500, 600, 700, 800, 900, 950, 960, or 963 nucleotides. In some embodiments, the sequence of the N-terminal hDSYF protein and the C-terminal hDSYF protein comprises an overlap of at least 50, 100, 150, 200, 250, 300 or 320 amino acids.

inverted end repetition

In some embodiments, polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions comprise, consist of, or consist essentially of one or more inverted terminal repeats (ITRS). It further includes a nucleotide sequence. In some embodiments, polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells and compositions comprise, consist of, or consist of 2, 3, 4, 5, 6 or more ITRs; and a sequence of 2, 3, 4, 5, 6 or more nucleotides consisting essentially of them. In some embodiments, two or more ITRs are the same. In some embodiments, two or more ITRs are different.

In some embodiments, a recombinant polynucleotide is flanked by two or more ITRs. In some embodiments, a 5' hDYSF polynucleotide is flanked by a first pair of ITRs. In some embodiments, the 3' hDYSF polynucleotide is flanked by a second pair of ITRs. In some embodiments, the ITRs within the first pair of ITRs are the same. In some embodiments, the ITRs within the first pair of ITRs are different. In some embodiments, the ITRs within the second pair of ITRs are the same. In some embodiments, the ITRs within the second pair of ITRs are different. In some implementations, the ITRs in the ITRs of the first pair are identical to the ITRs in the ITRs of the second pair. In some implementations, at least one ITR in the first pair of ITRs is identical to at least one ITR in the second pair of ITRs. In some implementations, the ITRs in the first pair of ITRs are different from the ITRs in the second pair of ITRs. In some implementations, at least one ITR in the first pair of ITRs is different from at least one ITR in the second pair of ITRs.

In some embodiments, the ITR is a viral ITR. In some embodiments, the ITR is an AAV ITR. In some embodiments, an AAV ITR is an AAV serotype AAVrh20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh74, AAV-8, AAV-9 , ITRs from at least one of AAV-10, AAVrh10, AAV-11, AAV-12 and AAV-13. In some embodiments, an AAV ITR is an AAV2 ITR. In some embodiments, the AAV ITR is an AAV5 ITR. ITR sequences for AAV1-6 can be found, for example, in Grimm et al., J Virol 80(1):426-39, 2006, which is incorporated by reference in its entirety.

In some embodiments, the recombinant polynucleotide does not contain AAV sequences other than inverted terminal repeats (ITRs).

In some embodiments, the recombinant polynucleotide contains no viral sequences other than inverted terminal repeats (ITRs).

In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the ITR is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO:3. In some embodiments, the ITR comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:3 over the entire length of SEQ ID NO:3. In some embodiments, the ITR comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO:3 over the entire length of SEQ ID NO:3. In some embodiments, the ITR comprises a nucleotide sequence comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less nucleotide mismatches with the nucleotide sequence of SEQ ID NO: 3 . In some embodiments, the ITR comprises a nucleotide sequence comprising 5 or fewer nucleotide mismatches with the nucleotide sequence of SEQ ID NO:3.

promoter

In some embodiments, the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise a nucleotide sequence comprising, consisting of, or consisting essentially of one or more promoters. . In some embodiments, the promoter is a eukaryotic promoter. Examples of eukaryotic promoters are the cytomegalovirus (CMV) promoter, elongation factor 1 alpha (EF1a) promoter, CAG promoter, phosphoglycerate kinase gene (PGK) promoter, tetracycline response element (TRE) promoter, human U6 nuclear (U6 ) promoter, and the UAS promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, a promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.

In some embodiments, the promoter is a tissue-specific promoter. Examples of tissues include, but are not limited to, muscle, epithelial, connective, and neural tissue. Examples of tissue-specific promoters are the B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, Desmin promoter, Elastase-1 promoter, Endoglin promoter, Fibronectin promoter, Flt-1 promoter, GFAP promoter, ICAM -2 promoter, INF-β promoter, Mb promoter, NphsI promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40/bAlb promoter, SV40/hAlb promoter, SV40/CD43 promoter, SV40/CD45 promoter , and the NSE/RU5' promoter.

In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is a myosin heavy chain complex-E box muscle creatine kinase fusion enhancer/promoter.

In some embodiments, the promoter is a recombinant promoter. In some embodiments, the recombinant promoter is a recombinant muscle-specific promoter. In some embodiments, the recombinant muscle-specific promoter is a recombinant myosin heavy chain-creatine kinase muscle-specific promoter. In another embodiment, the muscle-specific promoter is a human skeletal actin gene element, a cardiac actin gene element, a desmin promoter, a skeletal alpha-actin (ASKA) promoter, a troponin I (TNNI2) promoter, a myocyte-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid a-myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK7) promoter, C5-12 promoter, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factor.

In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:4. In some embodiments, the promoter is at least 80%, 82%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences.

intron

In some embodiments, polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise nucleotide sequences comprising, consisting of, or consisting essentially of one or more introns. . In some embodiments, an intron is a eukaryotic intron. In some embodiments, the intron is a mammalian intron. In some embodiments, an intron is a synthetic intron. In some embodiments, an intron is a chimeric intron. In some embodiments, introns are derived from non-coding exons. In some embodiments, the intron is upstream or 5' of the 5' hDYSF polynucleotide.

In some embodiments, an intron comprises at least one of a 5' donor site, branch point, or 3' splice site. In some embodiments, an intron comprises two or more of a 5' donor site, branch point, or 3' splice site. In some embodiments, an intron comprises a 5' donor site, a branch point, and a 3' splice site.

In some embodiments, the intron comprises a 5' donor region from the human β-globin gene.

In some embodiments, an intron comprises a branch point from an immunoglobulin G (IgG) heavy chain.

In some embodiments, an intron comprises a 3' splice acceptor site from an immunoglobulin G (IgG) heavy chain.

In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO:5. In some embodiments, the intron is at least 80%, 82%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences.

select marker

In some embodiments, the polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions further comprise a nucleotide sequence comprising, consisting of, or consisting essentially of one or more selectable markers. In some embodiments, the selectable marker is a bacterial selectable marker. In some embodiments, the selectable marker is an antibiotic resistance gene. Examples of antibiotic resistance genes include β-lactamase, kanamycin resistance gene, neo gene from Tn5, mutant FabI gene from E. coli genome, and URA3 (orotidine-5' phosphate decarboxylase from yeast) , but is not limited thereto. In some embodiments, the antibiotic resistance gene is a β-lactamase gene. In some embodiments, the antibiotic resistance gene is a kanamycin resistance gene.

polyadenylation signal

In some embodiments, polynucleotides, plasmids, viral vectors, vector systems, viral packaging systems, cells, and compositions comprise, consist of, or consist essentially of one or more polyadenylation (polyA) signals. It further contains the sequence. In some embodiments, the polyA signal is an artificial polyA signal.

In some embodiments, the polyA signal comprises the nucleotide sequence of SEQ ID NO:7. In some embodiments, the polyA signal is at least 80%, 82%, 85%, 87%, 90%, 92%, 93%, 94%, 95% of the nucleotide sequence of SEQ ID NO: 7 over the entire length of SEQ ID NO: 7. %, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences.

Expression Cassettes and Packaging Systems

Further disclosed herein are adeno-associated virus (AAV) expression cassettes. In some embodiments, an AAV expression cassette comprises (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any ITR disclosed herein; (b) any of the 5' hDYSF polynucleotides; and (c) a second ITR, wherein the second ITR comprises any ITR disclosed herein; wherein the 5' hDYSF polynucleotide of (b) is the first ITR of (a) and (c) above and flanked by a second ITR. In some embodiments, an AAV expression cassette comprising any of the 5' hDYSF polynucleotides disclosed herein is referred to as a 5' hDYSF AAV expression cassette.

Further disclosed herein are adeno-associated virus (AAV) plasmids. In some embodiments, an AAV expression cassette comprises (a) a first inverted terminal repeat (ITR), wherein the first ITR comprises any ITR disclosed herein; (b) any 3' hDYSF polynucleotide disclosed herein; and (c) a second ITR, wherein the second ITR comprises any ITR disclosed herein; wherein the 3' hDYSF polynucleotide of (b) is the first ITR of (a) and (c) above and flanked by a second ITR. In some embodiments, an AAV expression cassette comprising any 3' hDYSF polynucleotide disclosed herein is referred to as a 3' hDYSF AAV expression cassette.

In some embodiments, an adeno-associated virus (AAV) expression cassette comprises (a) a first inverted terminal repeat (ITR); (b) a polynucleotide sequence encoding a fragment of human dysferrin (hDYSF) protein, the polynucleotide sequence comprising: (i) the nucleotide sequence of SEQ ID NO: 1; (ii) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 of the nucleotide sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 13; (iv) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 of the nucleotide sequence of SEQ ID NO: 13 over the entire length of SEQ ID NO: 13; %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences; (v) a nucleotide sequence encoding a hDYSF protein, wherein the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or (vi) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v). %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences; and (c) a second ITR; wherein the polynucleotide sequence is flanked by first and second ITRs. In some embodiments, any AAV expression cassette disclosed herein further comprises one or more additional polynucleotide sequences comprising a promoter, intron, selectable marker, or origin of replication (ORI). In some embodiments, the AAV expression cassette comprises the nucleotide sequence of SEQ ID NO:6. In some embodiments, the AAV expression cassette is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6. %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the AAV expression cassette comprises the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the AAV expression cassette is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 15 over the entire length of SEQ ID NO: 15 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the AAV expression cassette does not further comprise a second polynucleotide sequence encoding a second fragment of hDYSF protein. In some embodiments, the AAV expression cassette contains no AAV sequences other than inverted terminal repeats (ITRs). In some embodiments, the AAV expression cassette contains no viral sequences other than inverted terminal repeats (ITRs).

In some embodiments, an adeno-associated virus (AAV) expression cassette comprises (a) a first inverted terminal repeat (ITR); (b) a polynucleotide sequence encoding a fragment of human dysferrin protein, wherein the polynucleotide sequence comprises: (i) the nucleotide sequence of SEQ ID NO: 2; (ii) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 of the nucleotide sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2; %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences; (iii) the nucleotide sequence of SEQ ID NO: 14; (ii) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 of the nucleotide sequence of SEQ ID NO: 14 over the entire length of SEQ ID NO: 14; %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences; (v) a polynucleotide sequence encoding a hDYSF protein, wherein the hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or (vi) at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of the polynucleotide sequence of (v) over the entire length of the nucleotide sequence of (v); 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences; and (c) a second ITR; wherein the polynucleotide sequence is flanked by first and second ITRs. In some embodiments, the AAV expression cassette comprises the nucleotide sequence of SEQ ID NO:8. In some embodiments, the AAV expression cassette is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 8 over the entire length of SEQ ID NO: 8. %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the AAV expression cassette comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the AAV expression cassette is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the nucleotide sequence of SEQ ID NO: 16 over the entire length of SEQ ID NO: 16 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences. In some embodiments, the AAV expression cassette further comprises one or more polynucleotide sequences comprising a selectable marker, origin of replication (ORI), untranslated region (UTR), or polyadenylation (polyA) signal.

Further disclosed herein is an adeno-associated virus (AAV) packaging system. In some embodiments, the AAV packaging system comprises (a) any of the 5' hDYSF AAV expression cassettes disclosed herein; (b) an adenovirus helper plasmid; and (c) rep-cap plasmid; In some embodiments, an adenovirus helper plasmid comprises one or more genes from an adenovirus. In some embodiments, one or more genes from adenovirus mediate AAV replication. In some embodiments, the one or more genes from adenovirus are selected from E4, E2a, and VA. In some embodiments, a rep-cap plasmid comprises one or more polynucleotides encoding adeno-associated virus rep and cap genes. In some embodiments, the rep gene encodes one or more of the life cycle proteins selected from Rep78, Rep68, Rep62, and Rep40. In some embodiments, the cap gene encodes one or more of the capsid proteins selected from VP1, VP2, and VP3. In some embodiments, a 5' hDYSF AAV expression cassette comprises one or more ITRs. In some embodiments, the ITR is an AAV ITR. In some embodiments, the serotype of the AAV ITR is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV ITR is different from the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is different from the serotype of the AAV capsid protein. In some embodiments, an AAV packaging system comprising any of the 5' hDYSF AAV expression cassettes disclosed herein is referred to as a 5' hDYSF AAV packaging system.

In some embodiments, the AAV packaging system comprises (a) any of the 3' hDYSF AAV expression cassettes disclosed herein; (b) an adenovirus helper plasmid; and (c) rep-cap plasmid; In some embodiments, an adenovirus helper plasmid comprises one or more genes from an adenovirus. In some embodiments, one or more genes from adenovirus mediate AAV replication. In some embodiments, the one or more genes from adenovirus are selected from E4, E2a, and VA. In some embodiments, a rep-cap plasmid comprises one or more polynucleotides encoding adeno-associated virus rep and cap genes. In some embodiments, the rep gene encodes one or more life cycle proteins selected from Rep78, Rep68, Rep62 and Rep40. In some embodiments, the cap gene encodes one or more of the capsid proteins selected from VP1, VP2, and VP3. In some embodiments, a 3' hDYSF AAV expression cassette comprises one or more ITRs. In some embodiments, the ITR is an AAV ITR. In some embodiments, the serotype of the AAV ITR is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV ITR is different from the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is the same as the serotype of the AAV capsid protein. In some embodiments, the serotype of the AAV rep gene is different from the serotype of the AAV capsid protein. In some embodiments, an AAV packaging system comprising any of the 3' hDYSF AAV expression cassettes disclosed herein is referred to as a 3' hDYSF AAV packaging system.

In some embodiments, the adeno-associated virus packaging system comprises (a) any of the 5' hDYSF AAV expression cassettes disclosed herein; and (b) an adenovirus helper plasmid. In some embodiments, the m adenovirus helper plasmid comprises one or more genes from an adenovirus. In some embodiments, one or more genes from adenovirus mediate AAV replication. In some embodiments, the one or more genes from adenovirus are selected from E4, E2a, and VA.

In some embodiments, the adeno-associated virus packaging system comprises (a) any of the 3' hDYSF AAV expression cassettes disclosed herein; and (b) an adenovirus helper plasmid. In some embodiments, an adenovirus helper plasmid comprises one or more genes from an adenovirus. In some embodiments, one or more genes from adenovirus mediate AAV replication. In some embodiments, the one or more genes from adenovirus are selected from E4, E2a, and VA.

virus vector

Further disclosed herein are adeno-associated virus (AAV) vectors (eg, AAV viruses or AAV particles). In some embodiments, an AAV vector comprises, consists of, or consists essentially of any 5' hDYSF polynucleotide disclosed herein. In some embodiments, an AAV vector comprising any of the 5' hDYSF polynucleotides disclosed herein is referred to as a 5' hDYSF AAV vector.

In some embodiments, a 5' hDYSF AAV vector comprises any of the 5' hDYSF AAV expression cassettes disclosed herein.

In some embodiments, an AAV vector comprises, consists of, or consists essentially of any 3' hDYSF polynucleotide disclosed herein. In some embodiments, an AAV vector comprising any 3' hDYSF polynucleotide disclosed herein is referred to as a 3' hDYSF AAV vector.

In some embodiments, a 3' hDYSF AAV vector comprises any of the 3' hDYSF AAV expression cassettes disclosed herein.

In some embodiments, the AAV vector is an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10, rh20, or rh74. In some embodiments, the AAV vector is AAV of serotype rh74. In some embodiments, the AAV vector is not serotype 5 AAV.

Further disclosed herein is a dual adeno-associated virus (AAV) vector system comprising two or more AAV vectors disclosed herein. In some embodiments, the dual AAV vector system comprises (a) a first AAV vector, wherein the first AAV vector comprises any of the 5' hDYSF polynucleotides disclosed herein; and (b) a second AAV vector, wherein the second AAV vector comprises any of the 3' hDYSF polynucleotides disclosed herein.

In some embodiments, the dual AAV vector system comprises (a) a first AAV vector, wherein the first AAV vector comprises, consists of, or consists essentially of any of the 5' hDYSF AAV vectors disclosed herein. configured; and (b) a second AAV vector, wherein the second AAV vector comprises, consists of, or consists essentially of any of the 3' hDYSF AAV vectors disclosed herein; be, or consist essentially of.

composition

Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the 5' hDYSF polynucleotides disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the 3' hDYSF polynucleotides disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the 5' hDYSF plasmids disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the 3' hDYSF plasmids disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the dual AAV vector systems disclosed herein. Further disclosed herein are compositions comprising, consisting of, or consisting essentially of any of the AAV vectors disclosed herein.

Further provided herein are (a) a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV comprises, consists of, or consists essentially of any of the 5' hDYSF polynucleotides disclosed herein; and (b) a pharmaceutically acceptable carrier, diluent, excipient or adjuvant;

Further provided herein are (a) a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV comprises, consists of, or consists essentially of any of the 3' hDYSF polynucleotides disclosed herein; and (b) a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

Further disclosed herein is (a) a first recombinant adeno-associated virus (rAAV), wherein the first rAAV comprises, consists of, or consists essentially of any of the 5' hDYSF polynucleotides disclosed herein. configured; and (b) a second recombinant adeno-associated virus (rAAV), wherein the second rAAV comprises, consists of, or consists essentially of any of the 3' hDYSF polynucleotides disclosed herein; A composition comprising, consisting of, or consisting essentially of is disclosed.

Further provided herein are (a) a first adeno-associated virus (AAV) particle, wherein the first AAV particle comprises, consists of, or is essentially of any of the 5' hDYSF AAV vectors disclosed herein. made up of; and (b) a second adeno-associated virus (AAV) particle, wherein the second AAV particle comprises, consists of, or consists essentially of any of the 3' hDYSF AAV vectors disclosed herein; Compositions comprising, consisting of, or consisting essentially of are disclosed.

In some embodiments, any of any of the compositions disclosed herein further comprises at least one of a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and preferably inert at the dosages and concentrations employed, and include buffers and surfactants such as pluronics. Examples of acceptable carriers include, but are not limited to, phosphate buffered saline, preservatives, and the like.

A pharmaceutically acceptable carrier, diluent or excipient may be suitable for injectable use. Examples of pharmaceutically acceptable carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg glycerol, propylene glycol, liquid polyethylene glycols, etc.), suitable mixtures thereof and vegetable oils. Proper fluidity can be maintained, for example, by the use of coatings, for example lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be caused by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be desirable to include a tonicity agent such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the polynucleotide, plasmid, viral vector, or dual vector system disclosed herein in the required amount in an appropriate solvent with various other ingredients, as enumerated above, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques which result in a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

AAV How to make a vector

Methods of producing adeno-associated virus (AAV) vectors (eg, viruses or viral particles) are disclosed herein. Methods for producing AAV vectors are known in the art. Such methods are disclosed, for example, in WO 01/83692, which is incorporated herein by reference in its entirety. General principles of AAV production are described, for example, in Carter, Current Opinions in Biotechnology 1533-1539, 1992; and "Muzyczka, Curr. Topics in Microbial and Immunol 158:97-129, 1992”, each of which is incorporated by reference in its entirety. Various approaches for producing AAV are described in Ratschin et al., Mol. Cell. Biol. 4:2072, 1984; "Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466, 1984; “Tratschin et al., Mol. Cell. Biol. 5:3251, 1985”; McLaughlin et al., J. Virol., 62:1963, 1988; and Lebkowski et al., Mol. Cell. Biol., 7:349, 1988; Samulski et al., J. Virol., 63:3822-3828, 1989; U.S. Patent No. 5,173,414; International Publication No. WO 95/13365; and corresponding U.S. Patent Nos. 5,658,776; International Publication No. WO 95/13392; WO 96/17947; International Application No. PCT/US98/18600; International Publication No. WO 97/09441 (International Application No. PCT/US96/14423); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; See Perrin et al., Vaccine 13:1244-1250, 1995; "Paul et al., Human Gene Therapy 4:609-615, 1993"; Clark et al., Gene Therapy 3:1124-1132, 1996; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. Each of these is incorporated by reference in its entirety.

In some embodiments, a method of producing an adeno-associated virus (AAV) vector comprises transducing a cell with any of the AAV packaging systems disclosed herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a recombinant cell that stably expresses adeno-associated virus rep and cap genes. In some embodiments, the method further comprises culturing the cells to produce a population of transduced cells. In some embodiments, the method further comprises collecting supernatant from the population of transduced cells. In some embodiments, the method further comprises subjecting the supernatant to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cellular debris and proteins. Alternatively, or additionally, the method further comprises lysing the population of transduced cells to produce a cell lysate. In some embodiments, the method further comprises subjecting the cell lysate to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cellular debris and protein. In some embodiments, the purity of the purified AAV vector sample is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

In some embodiments, a method of producing an adeno-associated virus (AAV) vector comprises transducing a cell with any of the 5' hDYSF AAV packaging systems disclosed herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a recombinant cell that stably expresses adeno-associated virus rep and cap genes. In some embodiments, the method further comprises culturing the cells to produce a population of transduced cells. In some embodiments, the method further comprises collecting supernatant from the population of transduced cells. In some embodiments, the method further comprises subjecting the supernatant to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cellular debris and protein. Alternatively, or additionally, the method further comprises lysing the population of transduced cells to produce a cell lysate. In some embodiments, the method further comprises subjecting the cell lysate to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cell debris and protein. In some embodiments, the purity of the purified AAV vector sample is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

In some embodiments, a method of producing an adeno-associated virus (AAV) vector comprises transducing a cell with any of the 3' hDYSF AAV packaging systems disclosed herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a recombinant cell that stably expresses adeno-associated virus rep and cap genes. In some embodiments, the method further comprises culturing the cells to produce a population of transduced cells. In some embodiments, the method further comprises collecting supernatant from the population of transduced cells. In some embodiments, the method further comprises subjecting the supernatant to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cellular debris and protein. Alternatively, or additionally, the method further comprises lysing the population of transduced cells to produce a cell lysate. In some embodiments, the method further comprises subjecting the cell lysate to one or more purification steps to produce a purified AAV vector sample, wherein the AAV vector sample is substantially free of cell debris and protein. In some embodiments, the purity of the purified AAV vector sample is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

cell

Further disclosed herein are cells comprising any of the 5' hDYSF polynucleotides disclosed herein. Cells may be prokaryotic or eukaryotic. Non-limiting examples of eukaryotic cells include mammalian, eg hamster, murine, rat, dog, sheep or human cells. In some embodiments, cells are transfected with a plasmid comprising any of the 5' hDYSF polynucleotides disclosed herein. In some embodiments, cells are transduced with any of the 5' hDYSF AAV expression cassettes disclosed herein. In some embodiments, cells are infected with any of the 5' hDYSF AAV vectors disclosed herein.

Further disclosed herein are cells comprising any of the 3' hDYSF polynucleotides disclosed herein. In some embodiments, cells are transfected with a plasmid comprising any of the 3' hDYSF polynucleotides disclosed herein. In some embodiments, cells are transduced with any of the 3' hDYSF AAV expression cassettes disclosed herein. In some embodiments, the cells are infected with any of the 3' hDYSF AAV vectors disclosed herein.

Any of the cells disclosed herein may be packaging cells that produce infectious rAAV. In some embodiments, the packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells and PerC6 cells (cognate 293 line). In another embodiment, the packaging cells are low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal cells) fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells). Non-limiting examples of prokaryotic cells include bacterial cells (eg Escherichia coli) and archaeal cells. Cells of the present disclosure can be used to produce a cell bank, such as an depository cell bank (ACB) or a GMP master cell bank (MCB) for non-GMP purposes. An aliquot of cells, in one embodiment, is expanded to a larger volume from the original inoculum prior to culture in a bioreactor for production.

treatment method

Further disclosed herein are methods of treating dysferinopathies. In some embodiments, a method of treating dysferinopathies comprises providing a subject in need thereof with (a) an effective amount of a first polynucleotide, wherein the first polynucleotide is any of the 5' hDYSF polynucleotides disclosed herein; and (b) an effective amount of a second polynucleotide, wherein the second polynucleotide is any of the 3' hDYSF polynucleotides disclosed herein. In some embodiments, the first polynucleotide is administered orally, parenterally (eg intramuscularly, intraperitoneally, intravenously, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasal, vaginal, rectal , sublingually, urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the first polynucleotide is administered intramuscularly or intravenously. In some embodiments, the second polynucleotide is administered orally, parenterally (e.g. intramuscular, intraperitoneal, intravenous, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasal, vaginal, rectal , sublingually, urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the second polynucleotide is administered intramuscularly or intravenously. In some embodiments, the first and second polynucleotides are administered simultaneously. In some embodiments, the first and second polynucleotides are administered sequentially. In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

In some embodiments, the method of treating dysferinopathy comprises administering to a subject in need thereof (a) an effective amount of a first adeno-associated virus (AAV) vector, wherein said first AAV vector is a 5' hDYSF disclosed herein; is any of the AAV vectors; and (b) an effective amount of a second adeno-associated virus (AAV) vector, wherein the second AAV vector is any of the 3' hDYSF AAV vectors disclosed herein; consists of this In some embodiments, the first AAV vector is administered orally, parenterally (eg intramuscularly, intraperitoneally, intravenously, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasal, vaginal, rectal , sublingually, urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the first AAV vector is administered intramuscularly or intravenously. In some embodiments, the second AAV vector is administered orally, parenterally (e.g. intramuscular, intraperitoneal, intravenous, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasal, vaginal, rectal , sublingually, urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the second AAV vector is administered intramuscularly or intravenously. In some embodiments, the first and second AAV vectors are administered simultaneously. In some embodiments, the first and second AAV vectors are administered sequentially. In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

In some embodiments, the method of treating dysferinopathy comprises administering to a subject in need thereof (a) an effective amount of a first AAV expression cassette, wherein the first AAV expression cassette is any of the 5' hDYSF AAV expression cassettes disclosed herein. belongs to; and (b) an effective amount of a second AAV expression cassette, wherein the second AAV expression cassette disclosed herein above is any of the 3' hDSYF AAV expression cassettes; do. In some embodiments, the first AAV expression cassette is administered orally, parenterally (eg intramuscularly, intraperitoneally, intravenously, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasally, vaginally, Administered by rectal, sublingual, urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the first AAV expression cassette is administered intramuscularly or intravenously. In some embodiments, the second AAV expression cassette is administered orally, parenterally (eg intramuscularly, intraperitoneally, intravenously, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasally, vaginally, Administered by rectal, sublingual, urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the second AAV expression cassette is administered intramuscularly or intravenously. In some embodiments, the first and second AAV expression cassettes are administered simultaneously. In some embodiments, the first and second AAV expression cassettes are administered sequentially. In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

In some embodiments, a method of treating dysferinopathies comprises administering to a subject in need thereof (a) any of the 5' hDYSF polynucleotides disclosed herein and any of the 3' hDYSF polynucleotides disclosed herein; (b) any of the 5' hDYSF AAV vectors disclosed herein and any of the 3' hDYSF AAV vectors disclosed herein; (c) any of the 5' hDYSF AAV expression cassettes disclosed herein and any of the 3' hDYSF AAV expression cassettes disclosed herein; or (d) any of the dual AAV vector systems disclosed herein; In some embodiments, the composition is administered orally, parenterally (eg intramuscular, intraperitoneal, intravenous, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasal, vaginal, rectal, sublingual , urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the composition is administered intramuscularly or intravenously. In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

Further herein, the use of any of the recombinant polynucleotides, plasmids, viral vectors, vector systems, and compositions in the manufacture of a medicament for treating dysferinopathy in a subject in need thereof is provided. is initiated As used herein, (a) any of the 5' hDYSF polynucleotides disclosed herein and the 3' any of the hDYSF polynucleotides; (b) any of the 5' hDYSF AAV vectors disclosed herein and any of the 3' hDYSF AAV vectors disclosed herein; (c) any of the 5' hDYSF AAV expression cassettes disclosed herein and any of the 3' hDYSF AAV expression cassettes disclosed herein; or (e) (d) any of the dual AAV vector systems disclosed herein. In some embodiments, the composition is administered orally, parenterally (e.g. intramuscular, intraperitoneal, intravenous, ICV, intrasternal injection or infusion, subcutaneous injection, or implant), inhalation spray nasal, vaginal, rectal, sublingual, Administered by urethral (eg urethral suppositories) or topical routes of administration (eg gels, ointments, creams, aerosols, etc.). In some embodiments, the composition is administered intramuscularly or intravenously. In some embodiments, the dysferinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

The titers of the AAV vectors to be administered in the methods of the present invention depend, for example, on the particular AAV, mode of administration, target of treatment, subject, and cell type(s) to be targeted, and are determined by standard methods in the art. can be determined The AAV titer per ml is at least about 1×10 ⁶ , about 1×10 ⁷ , about 1×10 ⁸ , about 1×10 ⁹ , about 1×10 ¹⁰ , about 1×10 ¹¹ , about 1×10 ¹² , about 1×10 ¹³ to about 1×10 ¹⁴ or more DNase resistant particles (DRPs). Also, the dosage can be expressed in units of viral genome (vg). For example, a dose of AAV may be at least about 1×10 ⁶ , about 1×10 ⁷ , about 1×10 ⁸ , about 1×10 ⁹ , about 1×10 ¹⁰ , about 1×10 ¹¹ , about 1×10 ¹² , about 2×10 ¹² , about 3×10 ¹² , about 4×10 ¹² , about 5×10 ¹² , about 6×10 ¹² , about 7×10 ¹² , about 8×10 ¹² , about 9×10 ¹² , about It may range from 1×10 ¹³ to about 1×10 ¹⁴ viral genomes.

AAV dosing can be performed by multiple assays including but not limited to ELISA, assessment of reverse transcriptase activity, FACS, transduction assay Northern blotting (eg semi-quantitative Northern), dot blot analysis or PCR (eg qPCR). method can be determined. It is well known that AAV dosage can be determined by measuring the AAV vector genome with quantitative real-time PCR (qPCR). This qPCR method overcomes inconsistent or random results from conventional transduction assays. In one embodiment of PCR dosage determination, plasmid DNA is used as a calibration standard. The form of the plasmid can affect dosage results from qPCR methods. In one embodiment, circular or supercoiled DNA or plasmids are used as quantification standards.

In some embodiments, dosages can be expressed in units of vg/kg, based on supercoiled DNA or plasmid as a quantification standard. For example, the dose of AAV may be about 1×10 6 -1×10 16 vg/kg, about 1×10 ⁸ to 1×10 ¹⁵ vg/kg, or about 1×10 15 vg/kg, based on supercoil DNA or plasmid as a quantification standard. 10 ¹⁰ to 1×10 ¹⁴ vg/kg. In another embodiment, the dosage is at least about 1×10 ⁶ , about 1×10 ⁷ , about 1×10 ⁸ , about 1×10 ⁹ , about 1×10 ¹⁰ , about 1×10 ¹¹ , about 1×10 ¹² , about 2×10 ¹² , about 4×10 ¹² , about 6×10 ¹² , about 8×10 ¹² , about 1×10 ¹³ , about 2×10 ¹³ , about 2.4×10 ¹³ , about 3×10 ¹³ , about 4×10 ¹³ , about 5×10 ¹³ , about 6×10 ¹³ , about 7×10 ¹³ , about 8×10 ¹³ , about 9×10 ¹³ , about 1×10 ¹⁴ , about 1×10 ¹⁵ , or at least It is about 1×10 ¹⁶ vg/kg. In one embodiment, the dose is at least 2×10 ¹² , 4×10 ¹² , 6×10 ¹² , 8×10 ¹² , 1×10 ¹³ , 2×10 ¹³ based on supercoil DNA or plasmid as a quantification standard. , 2.4×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , or 8×10 ¹³ vg/kg.

In some embodiments, a method disclosed herein can be used for at least about 1×10 ⁶ , about 1×10 ⁷ , about 1×10 ⁸ , about 1×10 ⁹ , about 1×10 ¹⁰ , about 1×10 ¹¹ , about 1× 10 ¹² , about 2×10 ¹² , about 3×10 ¹² , about 4×10 ¹² , about 5×10 ¹² , about 6×10 ¹² , about 7×10 ¹² , about 8×10 ¹² , about 9×10 ¹² , about 1×10 ¹³ vg in a total volume of 1.5 ml per injection. In some embodiments, a method disclosed herein can be used for at least about 1×10 ⁶ , about 1×10 ⁷ , about 1×10 ⁸ , about 1×10 ⁹ , about 1×10 ¹⁰ , about 1×10 ¹¹ , about 1× 10 ¹² , about 2×10 ¹² , about 3×10 ¹² , about 4×10 ¹² , about 5×10 ¹² , about 6×10 ¹² , about 7×10 ¹² , about 8×10 ¹² , about 9×10 ¹² , about 1×10 ¹³ , about 2×10 ¹³ , about 5×10 ¹³ , about 7×10 ¹³ , about 1×10 ¹⁴ vg total daily dose. One exemplary method for determining encapsidated vector genome titer is quantitative PCR, eg, Pozsgai et al., Mol. Ther. 25(4): 855-869, 2017”, which is incorporated by reference in its entirety.

In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein are administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times daily. dosed twice. In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein are administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 administrations. In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein are administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 times. In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein are administered to a subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. dosed every In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein can be administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , administered every 12, 13, or 14 weeks. In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein are administered to a subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. administered for a period of In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein can be administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks. In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein can be administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 months.

In some embodiments, the methods disclosed herein include systemic administration of any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein. Systemic administration, for example, is administration into the circulatory system such that the entire body is affected. Systemic administration includes enteral administration, eg absorption through the gastrointestinal tract and parenteral administration via injection, infusion or implantation.

In some embodiments, the methods disclosed herein include topically administering any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein. In some embodiments, the methods disclosed herein include administering any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein to one or more tissues. In some embodiments, the tissue is selected from muscle, epithelial, connective and neural tissue. In some embodiments, the tissue is muscle tissue.

In some embodiments, the methods disclosed herein include administering to a foot of a subject any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein. In some embodiments, the methods disclosed herein comprise administering any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein to an extensor brevis (EDB) muscle of a subject.

Combination therapies are also contemplated by the present invention. Combinations as used herein include both simultaneous and sequential treatments. Combinations of the methods of the present invention with standard medical treatments (eg corticosteroids) are specifically contemplated as combinations with novel therapies.

In some embodiments, the methods disclosed herein detect the presence or It further includes detecting the absence. In some embodiments, any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein are administered to a subject upon detection of the presence of a mutation in the dysferrin gene. 예시적인 디스페린 돌연변이는 c1392dupA, c3035GA (pW1012X), c2858dupT, c2779del G, c5594delG, c4201dupA, c1795_1799dupTACT, c3832CT (pQ1278X), c757CT (pR253W), c855+1delG, c3126GA (pW1042X), c1663CT (pR555W), c610CT ( pR204X), c3112CT (pR1038X), c1368CG (pC456W), c5713CT (pR1905X), c3826CG (pI1276V), c3843+1GA, c4167+1GC, c2643+1GA, c797TC (pI266P), c4876delG, c3477CA (pY1159X), c3137GA (pR1046H ), c509CA (pA170E), c3967CT (pQ1323X), 3191_3196dupGAGGCG, c3992GT (pR1331L), c3516_3517delTT, c247delG, c1180+11CT, c896GA (pG299E), c5078GA (pR1693Q), c5979dupA, c3348+1_3348+4delGTAT, c5314_5318delAGCCC, 및 c565CG ( pL189V), but is not limited thereto. 일부 경우에, 디스페린 유전자는 c1392dupA, c3035GA (pW1012X), c2858dupT, c2779delG, c5594delG, c4201dupA, c1795_1799dupTACT, c3832CT (pQ1278X), c757CT (pR253W), c855+1delG, c3126GA (pW1042X), c1663CT (pR555W), c610CT (pR204X), c3112CT (pR1038X), c1368CG (pC456W), c5713CT (pR1905X), c3826CG (pI1276V), c3843+1GA, c4167+1GC, c2643+1GA, c797TC (pI266P), c4876delG, c4876delG, c347 pR1046H), c509CA (pA170E), c3967CT (pQ1323X), 3191_3196dupGAGGCG, c3992GT (pR1331L), c3516_3517delTT, c247delG, c1180+11CT, c896GA (pG299E), c5078GA (pR1693Q), c5979dupA, c3348+1_3348+4delGTAT, c5314_5318delAGCCC, 및 c565CG (pL189V).

In some embodiments, a method disclosed herein comprises detecting the level of dysferrin protein in a subject prior to or after administering to the subject any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein. contains more In some embodiments, the methods disclosed herein further comprise detecting the level of dysferrin protein in the subject after administering to the subject any of the polynucleotides, plasmids, viral vectors, dual vector systems, or compositions disclosed herein. . In some embodiments, detecting the level of dysferrin comprises detecting expression of the dysferrin gene. Detecting expression of the dysferrin gene may include quantifying dysferrin DNA or RNA levels. Alternatively, or additionally, detecting the level of dysferrin protein includes quantifying the level of dysferrin protein. In some embodiments, the level of dysferrin protein is detected in a sample from the subject. In some embodiments, the sample is a bodily fluid sample. Examples of bodily fluid samples include, but are not limited to, blood, urine, sweat, saliva, feces, and synovial fluid. In some embodiments, the blood sample is a plasma or serum sample. In some cases, the method further comprises a dysferrin DNA sequencing test, eg, from Athena Diagnostics (CPT: 81408(1)).

In some embodiments, the methods disclosed herein further comprise modifying the dose or frequency of administration of any of the polynucleotides, plasmids, viral vectors, dual vector systems or compositions administered to the subject. In some embodiments, alteration of dosage or frequency of administration is based on detection of dysferrin protein levels. In some embodiments, the dose or frequency of administration is determined at an earlier time in the subject's dysferrin protein level (e.g., prior to administration of the polynucleotide, plasmid, viral vector, dual vector system, or composition, or to a polynucleotide, plasmid, virus increase compared to the dysferrin protein level in a subject after administration of an initial dose of the vector, dual vector system, or composition, but prior to administration of subsequent doses of the polynucleotide, plasmid, viral vector, dual vector system, or composition) is reduced when

kit

In another aspect, a kit comprising, or alternatively consisting essentially of, or further consisting of any one or more of polynucleotides, polypeptides, vectors, cells and systems, or compositions, and instructions for use is provided. Provided. In one aspect, any one or more of the polynucleotides, polypeptides, vectors, cells and systems, or compositions are detectably labeled or further comprise a purified or detectable marker. In some cases, the kit comprises: a) a first polynucleotide, wherein the first polynucleotide is a recombinant polynucleotide described herein, and a second polynucleotide, wherein the second polynucleotide is a recombinant polynucleotide described herein; or b) a first adeno-associated virus (AAV) vector, wherein said first AAV vector is an AAV vector described herein, and a second adeno-associated virus (AAV) vector, wherein said second AAV vector is an AAV vector described herein. is a vector; or c) an AAV dual vector system described herein; or d) a composition described herein; or e) a cell described herein (eg a host cell, optionally a mammalian cell); and, optionally, instructions for use.

Example

Example 1: double AAV Creation of vector systems

This example provides an exemplary method for producing the dual AAV vector system disclosed herein. In this example, a double AAV vector, rAAVrh74.MHCK7.DYSF.DV, is produced. rAAVrh.74.MHCK7.DYSF.DV is a non-cloning recombinant AAV serotype rh74 (AAVrh74) that expresses human dysferrin from a dual vector (DV) under the control of the muscle specific MHCK7 promoter. The dual vector contains either the 5' or 3' portions of the dysferrin cDNA sequence, and these portions overlap by -1 kb to facilitate recombination and create the full-length human dysferrin gene. An expression cassette containing a portion of the human dysferrin cDNA is flanked by AAV2 inverted terminal repeat sequences (ITRs) (FIG. 1).

To construct rAAVrh.74.MHCK7.DYSFDV, human dysferin cDNA was split into two constructs attached to the packaging capacity of AAV (4.7 kb). The 5' vector (e.g. 5' hDYSF AAV vector), pAAV.MHCK7.DYSF5'.PTG (PTG=promoter/transgene) contains the muscle-specific MHCK7 promoter, chimeric intron, consensus Kozak sequence and the amino acids of the dysferrin amino acid sequence. It contains the 5' portion of the DYSF cDNA corresponding to 1-1113. A 3' vector (e.g. 3' hDYSF AAV vector), pAAV.DYSF3'.POLYA, is a DYSF 3'UTR harboring a polyadenylation signal and DYSF corresponding to amino acids 794-2080 of the disferrin amino acid sequence contains the 3' portion of cDNA. The sequences of the expression cassettes of the 5' hDYSF AAV vector and the 3' hDYSF AAV vector are disclosed as SEQ ID NOs: 6 and 8, respectively.

Previous studies have demonstrated cardiac expression using the MHCK7 promoter (Salva et al., Mol Ther 15, 320-329 (2007), incorporated by reference in its entirety) and AAVrh.74 to achieve skeletal, diaphragmatic, and myocardial expression. It was achieved ("Sondergaard et al., Annals of Clinical and Transl Neurology 2, 256-270 (2015)", which is incorporated by reference in its entirety). The 5' hDYSF AAV vector and the 3' hDSYF AAV vector were encapsidated into separate AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from rhesus macaque lymph nodes and is discussed in Rodino-Klapac et al., Journal of Translational medicine 5, 45 (2007), which is incorporated by reference in its entirety.

5' hDYSF AAV vector( AAV vector plasmid pAAV . MHCK7. DYSF5 '. PTG )

A first recombinant single-stranded AAV vector was generated using the AAV vector DNA plasmid pAAV.MHCK7.DYSF5'.PTG. Plasmid construction by inserting the MHCK7 expression cassette driving the 5' portion of the human dysferrin partial cDNA sequence (human cDNA, Genbank Accession No. NM_003494.3) into the vector backbone pAAV-CMV (Clontech) (see Figure 2 for plasmid map and Table 1 for specific sequence information). A chimeric intron exists, and the 5' donor site from the first intron of the human β-globin gene and the branch point and 3' spleen from the intron between the body and the leader of the heavy chain variable region of the immunoglobulin gene Consists of the Rice receptor site. The only viral sequence contained in this vector is the inverted terminal repeat of AAV2 required for both viral DNA replication and packaging of the rAAV vector genome. The sequence between the two ITRs is the portion of DNA encapsidated into the AAVrh.74 virion.

[Table 1]

3' hDYSF AAV vector( AAV vector plasmid pAAV . DYSF3 '. POLY )

A second recombinant single-stranded AAV vector was produced using the AAV vector DNA plasmid pAAV.DYSF3'.POLYA. A plasmid was constructed by inserting the human dysferrin partial cDNA sequence (human cDNA, Genbank Accession No. NM_003494.3) into the vector backbone pAAV-CMV (Clontech) (see Figure 3 for plasmid map and Table 2 for specific sequence information). ). Endogenous dysferrin 3' untranslated region and polyA signal sequence were used for efficient transcription termination. The only viral sequence contained in this vector is the inverted terminal repeat of AAV2 required for both viral DNA replication and packaging of the rAAV vector genome. The sequence between the two ITRs is the portion of DNA encapsidated into the AAVrh.74 virion.

[Table 2]

AAV helper plasmid ( pNLRep2 - Caprh74 )

The parent plasmid, pNLrep, was constructed from p5E18 and pCLR3K. [Document "Bansal, D. , et al., Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 423 , 168-172 (2003), which is incorporated by reference in its entirety]. p5E18 is based on pAAV/Ad. It contains the AAV2 rep and cap genes, the p5 promoter is removed from the 5' end of rep and placed at the 3' end of cap, which results in the presence of a 3 kb spacer sequence between p5 and rep (specific sequence information see Table 3). To generate pCLR3K, a human collagen intron was amplified by PCR and then cloned into pAd/AAV at position 1.052. To construct pNLrep, the BamHI/XbaI fragment of p5E18 was replaced with a BamHI/XbaI fragment containing the 3 kb collagen intron from pCLR3K. The rh74 cap gene was PCR amplified and cloned in place of the AAV2 cap gene in pNLrep using Swa I/Not I restriction sites to obtain pNLRep2-Caprh74. The identity of the AAV rh74 capsid gene was confirmed by DNA plasmid sequencing.

[Table 3]

adenovirus helper Plasmid ( pHELP )

Plasmid pHELP was obtained from Applied Viromics (Fremont, CA 94538) and is 11,635 bp in size (see Table 4 for specific sequence information). The plasmid contains regions of the adenovirus genome important for AAV replication, namely the E2A, E4 and VA RNAs (adenovirus E1 functions are provided by 293 cells). The plasmid is based on the pBluescript backbone, and also confers resistance to ampicillin (10,182-11,042 bp), the bacterial ColE1 origin of replication (9,315-10,167 bp) and the f1 single-stranded DNA origin of replication (11,172-11,627 bp). It contains the bla gene encoding the TEM-1 β-lactamase gene. The adenoviral sequences present on this plasmid represent only about 28% (9,280 / 35,938) of the adenovirus genome and do not contain cis elements important for replication, such as inverted terminal repeats. The identity of these three adenovirus genes was confirmed by DNA plasmid sequencing performed. DNA analysis showed 100% homology to three adenovirus type 5 gene regions (GenBank accession number AF369965, incorporated by reference in its entirety).

[Table 4]

Example 2: double AAV Preparation of viral products using vector systems

HEK 293 cells were transfected with three production plasmids ((i) an AAV vector plasmid, e.g., a 5' hDYSF AAV vector or a 3' hDYSF AAV vector; (ii) an adenovirus (Ad) helper. plasmid; and (iii) an AAV helper plasmid). Transfecting the cells involved preparing a DNA/calcium solution containing AAV vector plasmid, Ad helper plasmid, AAV helper plasmid and CaCl ₂ and mixing with an equal volume of 2X HEPES buffered saline to obtain an optimal precipitate. The precipitate was then added to HEK 293 cells and incubated. The precipitate was then added to HEK 293 cells and incubated. After culturing, the medium was exchanged, and at this time, nuclease was added.

Example 3: rAAVrh . 74.MHCK7.DYSF . DV intramuscular Determining the efficacy of delivery

Two AAV expression cassettes were generated containing the 5' and 3' portions of the MHCK7.DYSF cassette with ~1 kb of overlapping sequence (see Figure 1). The plasmid was packaged into an AAVrh74 vector. 4-week-old Dysf ^-/- mice were treated with 1×10 ¹¹ vg of each vector by intramuscular injection into the tibialis anterior muscle and necropsied at 1 month. Robust full-length dysferrin expression was shown after delivery of both vectors by immunostaining (FIG. 4A) and Western blot (FIG. 4C). Delivery of either vector alone did not result in aberrant dysferrin expression (FIG. 4B, immunostaining, and FIG. 4D, Western blot). 3222 is the full-length control. The number of myofibers expressing dysferrin was quantified and shown in Table 5.

[Table 5]

A time course study was initiated to evaluate safety after intramuscular injection into the tibialis anterior muscle (Table 6). Protein expression and vector biodistribution were also evaluated. At the 1, 3, 6, 9, and 12 month endpoints, animals were thoroughly necropsied and dysferin expression on muscle and non-target organs (Figures 5A-5C (1, 3, and 6 months indicated), vector biopsy Analysis of vector biodistribution (Table 7) and histopathology.Tibialis muscle (TA) was injected.Tissues analyzed for histopathology for each animal included: gonads, liver, heart , lung, spleen, kidney, diaphragm, left (treated) and right tibialis anterior muscle.No finding was confirmed.

[Table 6]

[Table 7]

After intramuscular injection, expression of dysferin was found in the injected tibialis anterior muscle (see Figure 6). After intravenous delivery, expression of dysferrin was found in skeletal and cardiac muscle (see FIG. 7A ).

Additional cohorts of mice were treated to determine the minimum effective dose for membrane repair. Three doses of AAV vectors were injected into the FDB of 8-week-old 129Dysf−/− mice (n=6 per group). A control Dysf-/- group received saline, and a group of 129WT mice served as strain-specific normal controls. As shown in Figure 8, AAVrh.74.DYSF.DV treatment showed dose dependent membrane resealing. Parallel expression studies show that high doses result in >50% expression of fiber transduction. This dose, when normalized to muscle weight, is equivalent to that given to the tibialis anterior muscle for expression and safety studies (see FIGS. 4A-5C ).

[Table 8]

rAAVrh . 74.MHCK7.DYSF . DV's systemic delivery

A dose finding study was conducted to test the feasibility/effectiveness of systemic delivery of dual vector delivery. BlaJ mice (AJ dysferrin deficient mice backcrossed to BL6 mice) were used for studies based on functional MRI/MRS results established in this strain. Three groups of mice (n=6 per group) were injected at 6 weeks of age with saline, 2e12 vg total AAV.DYSF DV (8e13 vg/kg based on supercoil DNA or plasmid as quantification standard), or 6e12 vg total AAV.DYSF DV ( 2.4e13 vg/kg) based on supercoiled DNA or plasmid as quantification standard) by tail vein injection. Endpoint analyzes were performed at 3 months and included diaphragm physiology, analysis of membrane repair of FDB, and complete necropsies to quantify dysferin expression and assess histopathology. ⁴

At the study endpoint of 4 months, a complete necropsy was performed. The diaphragm was force measured, FDB muscle was tested for restoration of membrane repair, and muscle and organs were harvested for expression, vector biodistribution and histopathology. At high doses, dysferrin expression was extensive in all muscles (Figures 7A and 7B), whereas low doses had low levels of variable expression. At 20 weeks, BlaJ has a histologically very mild phenotype. There is a significant increase in the central nucleus as a marker of regeneration compared to control. Treated mice showed a significant reduction in central nucleation at high doses (FIG. 7B). The most affected muscle, the psoa, showed reduced fibrosis and inflammation when treated at high doses (6e12 vg) (FIG. 9). The diaphragmatic force deficit was restored at both high and low doses (FIG. 10A), and there was a dose response in membrane repair of the FDB muscle (FIG. 10B).

Example 4: in the NHP AAV5 and Aavrh . 74. Mhck7. Dysf . Dv Safety and Efficacy of Full-Length Dysferin Expression by Delivery

Methods: Four NHPs were treated with AAV.MHCK7.DYSF.FLAG by intramuscular injection. Two NHPs were treated with AAV5.MHCK7.DYSF and two with AAVrh.74.MHCK7.DYSF.FLAG. Reflecting our clinical trial design, one animal was analyzed at 3 months and two at 6 months. Animals underwent basic chemistry and immunology studies including ELISpot assays to measure T cells for AAV5 and rh.74 capsid and dysferin (FIGS. 11A-11D) and anti-AAV Ab titers (Table 9).

Peptide pools used to stimulate PBMCs were designed to be 15 amino acids long with an overlap of 10 amino acids to capture all possible antigenic epitopes. Cells responding to the peptide release interferon-γ quantified as spots via ELISpot assay. Spots per million cells were counted as 50 spots/1×10 ⁶ cells as a positive reaction threshold. No sustained immune response was observed. All animals had expression at the end of the study (FIG. 16A). These studies were repeated every 2 weeks for the entire study. At study endpoint, a full necropsy was performed on the animals, which included histopathology and biodistribution studies on living organ tissues in addition to gene expression studies.

Results: No observable toxicity was found. Applicants used an anti-dysferin antibody that does not discriminate between rhesus and human dysferin to demonstrate overexpression of dysferin (FIGS. 12a to 12c). In addition, anti-FLAG immunostaining was performed to confirm expression of vector-derived dysferin (FIG. 13). For AAV5.DYSF injected TA, muscle showed 104.9% (3mo) and 122.6% (6mo) overexpression of dysferin, whereas AAVrh74.DYSF.DV injected TA showed 122.0% (3mo) overexpression of dysferin compared to non-injected controls. ) and 115.2% 6mo) overexpression. No toxicity was observed at the tissue level in NHPs lacking inflammation or myofibrillar necrosis. Immunological analysis did not reveal any abnormal response to the capsid or transgene by ELISpot (Figs. 11a to 11d). Also, sufficiently complete blood counts and chemistry panels did not show abnormal values in any macaques. As expected, anti-AAV antibody titers were elevated after gene transfer. Endpoint anti-AAVrh74 titers were lower than those for anti-AAV5.

[Table 9]

equivalent

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing" and the like are to be read inclusively and without limitation. Further, the terms and expressions used herein are used for purposes of description and not limitation, and the use of such terms and expressions to exclude equivalents of the features shown and described or portions thereof is prohibited. Without intent, it is recognized that various modifications are possible within the scope of the claimed subject matter.

Accordingly, it should be understood that the materials, methods, and examples provided herein represent preferred embodiments, are illustrative, and are not intended as limitations on the scope of the present technology.

The technology has been broadly and comprehensively described herein. Each narrower species and sub-universal group falling within the general disclosure also forms part of the present description. This includes general descriptions of the subject matter with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Further, where a feature or embodiment of the present technology is described in terms of a Markush group, one skilled in the art will recognize that the present technology is also described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned in this specification are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

Other implementations are presented in the following claims.

SEQUENCE LISTING <110> RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL <120> GENE THERAPY WITH DYSFERLIN DUAL VECTORS <130> 106887-8070 <140> <141> <150> 63/024,338 <151> 2020-05-13 <160> 22 <170> PatentIn version 3.5 <210> 1 <211> 3340 <212> DNA <213> Homo sapiens <220> <223> 5' hDYSF DNA fragment v1 <400> 1 atgctgaggg tcttcatcct ctatgccgag aacgtccaca cacccgacac cgacatcagc 60 gatgcctact gctccgcggt gtttgcaggg gtgaagaaga gaaccaaagt catcaagaac 120 agcgtgaacc ctgtatggaa tgagggattt gaatgggacc tcaagggcat ccccctggac 180 cagggctctg agcttcatgt ggtggtcaaa gaccatgaga cgatggggag gaacaggttc 240 ctgggggaag ccaaggtccc actccgagag gtcctcgcca cccctagtct gtccgccagc 300 ttcaatgccc ccctgctgga caccaagaag cagccacag gggcctcgct ggtcctgcag 360 gtgtcctaca caccgctgcc tggagctgtg cccctgttcc cgccccctac tcctctggag 420 ccctccccga ctctgcctga cctggatgta gtggcagaca caggaggaga ggaagacaca 480 gaggaccagg gactcactgg agatgaggcg gagccattcc tggatcaaag cggaggcccg 540 ggggctccca ccaccccaag gaaactacct tcacgtcctc cgccccacta ccccgggatc 600 aaaagaaagc gaagtgcgcc tacatctaga aagctgctgt cagacaaacc gcaggatttc 660 cagatcaggg tccaggtgat cgaggggcgc cagctgccgg gggtgaacat caagcctggg 720 gtcaaggtta ccgctgcagg gcagaccaag cggacgcgga tccacaaggg aaacagccca 780 ctcttcaatg agactctttt cttcaacttg tttgactctc ctggggagct gtttgatgag 840 cccatcttta tcacggtggt agactctcgt tctctcagga cagatgctct cctcggggag 900 ttccggatgg acgtgggcac catttacaga gagccccggc acgcctatct caggaagtgg 960 ctgctgctct cagaccctga tgacttctct gctggggcca gaggctacct gaaaacaagc 1020 ctttgtgtgc tggggcctgg ggacgaagcg cctctggaga gaaaagaccc ctctgaagac 1080 aaggaggaca ttgaaagcaa cctgctccgg cccacaggcg tagccctgcg aggagcccac 1140 ttctgcctga aggtcttccg ggccgaggac ttgccgcaga tggacgatgc cgtgatggac 1200 aacgtgaaac agatctttgg cttcgagagt aacaagaaga acttggtgga cccctttggg 1260 gaggtcagct ttgcgggggaa aatgctgtgc agcaagatct tggagaagac ggccaaccct 1320 cagtggaacc agaacatcac actgcctgcc atgtttccct ccatgtgcga aaaaatgagg 1380 attcgtatca tagactggga ccgcctgact cacaatgaca tcgtggctac cacctacctg 1440 agtatgtcga aaatctctgc ccctggagga gaaatagaag aggagcctgc aggtgctgtc 1500 aagccttcga aagcctcaga cttggatgac tacctgggct tcctccccac ttttgggccc 1560 tgctacatca acctctatgg cagtcccaga gagttcacag gcttcccaga cccctacaca 1620 gagctcaaca caggcaaggg ggaagggtg gcttatcgtg gccggcttct gctctccctg 1680 gagaccaagc tggtggagca cagtgaacag aaggtggagg accttcctgc ggatgacatc 1740 ctccgggtgg agaagtacct taggaggcgc aagtactccc tgtttgcggc cttctactca 1800 gccaccatgc tgcaggatgt ggatgatgcc atccagtttg aggtcagcat cgggaactac 1860 gggaacaagt tcgacatgac ctgcctgccg ctggcctcca ccactcagta cagccgtgca 1920 gtctttgacg ggtgccacta ctactaccta ccctggggta acgtgaaacc tgtggtggtg 1980 ctgtcatcct actgggagga catcagccat agaatcgaga ctcagaacca gctgcttggg 2040 attgctgacc ggctggaagc tggcctggag caggtccacc tggccctgaa ggcgcagtgc 2100 tccacggagg acgtggactc gctggtggct cagctgacgg atgagctcat cgcaggctgc 2160 agccagcctc tgggtgacat ccatgagaca ccctctgcca cccacctgga ccagtacctg 2220 taccagctgc gcacccatca cctgagccaa atcactgagg ctgccctggc cctgaagctc 2280 ggccacagtg agctccctgc agctctggag caggcggagg actggctcct gcgtctgcgt 2340 gccctggcag aggagcccca gaacagcctg ccggacatcg tcatctggat gctgcaggga 2400 gacaagcgtg tggcatacca gcgggtgccc gcccaccaag tcctcttctc ccggcggggt 2460 gccaactact gtggcaagaa ttgtgggaag ctacagacaa tctttctgaa atatccgatg 2520 gagaaggtgc ctggcgcccg gatgccagtg cagatacggg tcaagctggg gtttgggctc 2580 tctgtggatg agaaggagtt caaccagttt gctgagggga agctgtctgt ctttgctgaa 2640 acctatgaga acgagactaa gttggccctt gttgggaact ggggcacaac gggcctcacc 2700 taccccaagt tttctgacgt cacgggcaag atcaagctac ccaaggacag cttccgcccc 2760 tcggccggct ggacctgggc tggagattgg ttcgtgtgtc cggagaagac tctgctccat 2820 gacatggacg ccggtcacct gagcttcgtg gaagaggtgt ttgagaacca gacccggctt 2880 cccggaggcc agtggatcta catgagtgac aactacaccg atgtgaacgg ggagaaggtg 2940 cttcccaagg atgacattga gtgcccactg ggctggaagt gggaagatga ggaatggtcc 3000 acagacctca accgggctgt cgatgagcaa ggctgggagt atagcatcac catccccccg 3060 gagcggaagc cgaagcactg ggtccctgct gagaagatgt actacacaca ccgacggcgg 3120 cgctgggtgc gcctgcgcag gagggatctc agccaaatgg aagcactgaa aaggcacagg 3180 caggcggagg cggagggcga gggctggggag tacgcctctc tttttggctg gaagttccac 3240 ctcgagtacc gcaagacaga tgccttccgc cgccgccgct ggcgccgtcg catggagcca 3300 ctggagaaga cggggcctgc agctgtgttt gcccttgagg 3340 <210> 2 <211> 3866 <212> DNA <213> Homo sapiens <220> <223> 3' hDYSF DNA fragment v1 <400> 2 tcgtcatctg gatgctgcag ggagacaagc gtgtggcata ccagcgggtg cccgcccacc 60 aagtcctctt ctcccggcgg ggtgccaact actgtggcaa gaattgtggg aagctacaga 120 caatctttct gaaatatccg atggagaagg tgcctggcgc ccggatgcca gtgcagatac 180 gggtcaagct gtggtttggg ctctctgtgg atgagaagga gttcaaccag tttgctgagg 240 ggaagctgtc tgtctttgct gaaacctatg agaacgagac taagttggcc cttgttggga 300 actggggcac aacgggcctc acctacccca agttttctga cgtcacgggc aagatcaagc 360 tacccaagga cagcttccgc ccctcggccg gctggacctg ggctggagat tggttcgtgt 420 gtccggagaa gactctgctc catgacatgg acgccggtca cctgagcttc gtggaagagg 480 tgtttgagaa ccagacccgg cttcccggag gccagtggat ctacatgagt gacaactaca 540 ccgatgtgaa cggggagaag gtgcttccca aggatgacat tgagtgccca ctgggctgga 600 agtgggaaga tgaggaatgg tccacagacc tcaaccgggc tgtcgatgag caaggctggg 660 agtatagcat caccatcccc ccggagcgga agccgaagca ctgggtccct gctgagaaga 720 tgtactacac acaccgacgg cggcgctggg tgcgcctgcg caggagggat ctcagccaaa 780 tggaagcact gaaaaggcac aggcaggcgg aggcggaggg cgagggctgg gagtacgcct 840 ctctttttgg ctggaagttc cacctcgagt accgcaagac agatgccttc cgccgccgcc 900 gctggcgccg tcgcatggag ccactggaga agacggggcc tgcagctgtg tttgcccttg 960 agggggccct gggcggcgtg atggatgaca agagtgaaga ttccatgtcc gtctccacct 1020 tgagcttcgg tgtgaacaga cccacgattt cctgcatatt cgactatggg aaccgctacc 1080 atctacgctg ctacatgtac caggcccggg acctggctgc gatggacaag gactcttttt 1140 ctgatcccta tgccatcgtc tccttcctgc accagagcca gaagacggtg gtggtgaaga 1200 acacccttaa ccccacctgg gaccagacgc tcatcttcta cgagatcgag atctttggcg 1260 agccggccac agttgctgag caaccgccca gcattgtggt ggagctgtac gaccatgaca 1320 cttatggtgc agacgagttt atgggtcgct gcatctgtca accgagtctg gaacggatgc 1380 cacggctggc ctggttccca ctgacgaggg gcagccagcc gtcgggggag ctgctggcct 1440 cttttgagct catccagaga gagaagccgg ccatccacca tattcctggt tttgaggtgc 1500 aggagacatc aaggatcctg gatgagtctg aggacacaga cctgccctac ccaccacccc 1560 agagggaggc cacacatctac atggttcctc agaacatcaa gccagcgctc cagcgtaccg 1620 ccatcgagat cctggcatgg ggcctgcgga acatgaagag ttaccagctg gccaacatct 1680 cctcccccag cctcgtggta gagtgtgggg gccagacggt gcagtcctgt gtcatcagga 1740 acctccggaa gaaccccaac tttgacatct gcaccctctt catggaagtg atgctgccca 1800 gggaggagct ctactgcccc cccatcaccg tcaaggtcat cgataaccgc cagtttggcc 1860 gccggcctgt ggtgggccag tgtaccatcc gctccctgga gagcttcctg tgtgacccct 1920 actcggcgga gagtccatcc ccacagggtg gcccagacga tgtgagccta ctcagtcctg 1980 gggaagacgt gctcatcgac attgatgaca aggagcccct catcccccatc caggaggaag 2040 agttcatcga ttggtggagc aaattctttg cctccatagg ggagagggaa aagtgcggct 2100 cctacctgga gaaggatttt gacaccctga aggtctatga cacacagctg gagaatgtgg 2160 aggcctttga gggcctgtct gacttttgta acaccttcaa gctgtaccgg ggcaagacgc 2220 aggagggagac agaagatcca tctgtgattg gtgaatttaa gggcctcttc aaaatttatc 2280 ccctcccaga agacccagcc atccccatgc ccccaagaca gttccaccag ctggccgccc 2340 agggacccca ggaggtgcttg gtccgtatct acattgtccg agcatttggc ctgcagccca 2400 aggaccccaa tggaaagtgt gatccttaca tcaagatctc catagggaag aaatcagtga 2460 gtgaccagga taactacatc ccctgcacgc tggagcccgt atttggaaag atgttcgagc 2520 tgacctgcac tctgcctctg gagaaggacc taaagatcac tctctatgac tatgacctcc 2580 tctccaagga cgaaaagatc ggtgagacgg tcgtcgacct ggagaacagg ctgctgtcca 2640 agtttggggc tcgctgtgga ctcccacaga cctactgtgt ctctggaccg aaccagtggc 2700 gggaccagct ccgcccctcc cagctcctcc acctcttctg ccagcagcat agagtcaagg 2760 cacctgtgta ccggacagac cgtgtaatgt ttcaggataa agaatattcc attgaagaga 2820 tagaggctgg caggatccca aacccacacc tgggcccagt ggaggagcgt ctggctctgc 2880 atgtgcttca gcagcagggc ctggtcccgg agcacgtgga gtcacggccc ctctacagcc 2940 ccctgcagcc agacatcgag caggggaagc tgcagatgtg ggtcgaccta tttccgaagg 3000 ccctggggcg gcctggacct cccttcaaca tcaccccacg gagagccaga aggtttttcc 3060 tgcgttgtat tatctggaat accagagatg tgatcctgga tgacctgagc ctcacggggg 3120 agaagatgag cgacatttat gtgaaaggtt ggatgattgg ctttgaagaa cacaagcaaa 3180 agacagacgt gcattatcgt tccctgggag gtgaaggcaa cttcaactgg aggttcattt 3240 tccccttcga ctacctgcca gctgagcaag tctgtaccat tgccaagaag gatgccttct 3300 ggaggctgga caagactgag agcaaaatcc cagcacgagt ggtgttccag atctggggaca 3360 atgacaagtt ctcctttgat gattttctgg gctccctgca gctcgatctc aaccgcatgc 3420 ccaagccagc caagacagcc aagaagtgct ccttggacca gctggatgat gctttccacc 3480 cagaatggtt tgtgtccctt tttgagcaga aaacagtgaa gggctggtgg ccctggtgtag 3540 cagaagaggg tgagaagaaa atactggcgg gcaagctgga aatgaccttg gagattgtag 3600 cagagagtga gcatgaggag cggcctgctg gccagggccg ggatgagccc aacatgaacc 3660 ctaagcttga ggacccaagg cgccccgaca cctccttcct gtggtttacc tccccataca 3720 agaccatgaa gttcatcctg tggcggcgtt tccggtgggc catcatcctc ttcatcatcc 3780 tcttcatcct gctgctgttc ctggccatct tcatctacgc cttcccgaac tatgctgcca 3840 tgaagctggt gaagcccttc agctga 3866 <210> 3 <211> 128 <212> DNA <213> Adeno-associated virus 2 <220> <223> ITR <400> 3 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttcct 128 <210> 4 <211> 792 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> promoter <400> 4 aagcttgcat gtctaagcta gacccttcag attaaaaata actgaggtaa gggcctgggt 60 aggggaggtg gtgtgagacg ctcctgtctc tcctctatct gcccatcggc cctttgggga 120 ggaggaatgt gcccaaggac taaaaaaagg ccatggagcc agaggggcga gggcaacaga 180 cctttcatgg gcaaaccttg gggccctgct gtctagcatg ccccactacg ggtctaggct 240 gcccatgtaa ggaggcaagg cctggggaca cccgagatgc ctggttataa ttaacccaga 300 catgtggctg cccccccccc cccaacacct gctgcctcta aaaataaccc tgtccctggt 360 ggatcccctg catgcgaaga tcttcgaaca aggctgtggg ggactgaggg caggctgtaa 420 caggcttggg ggccagggct tatacgtgcc tgggactccc aaagtattac tgttccatgt 480 tcccggcgaa gggccagctg tccccccgcca gctagactca gcacttagtt taggaaccag 540 tgagcaagtc agcccttggg gcagcccata caaggccatg gggctgggca agctgcacgc 600 ctgggtccgg ggtgggcacg gtgcccgggc aacgagctga aagctcatct gctctcaggg 660 gcccctccct ggggacagcc cctcctggct agtcacaccc tgtaggctcc tctatataac 720 ccaggggcac aggggctgcc ctcattctac caccacctcc acagcacaga cagacactca 780 ggagcagcca gc 792 <210> 5 <211> 148 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> Intron <400> 5 aggtaagttt agtctttttg tctttattt caggtcccgg atccggtggt ggtgcaaatc 60 aaagaactgc tcctcagtgg atgttgcctt tacttctagg cctgtacgga agtgttactt 120 ctgctctaaa agctgcggaa ttgtaccc 148 <210> 6 <211> 4689 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> DYSF F5' Expression Cassette v1 <400> 6 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttccttg tagttaatga ttaacccgcc atgctctaga gtttaagctt gcatgtctaa 180 gctagaccct tcagattaaa aataactgag gtaagggcct gggtagggga ggtggtgtga 240 gacgctcctg tctctcctct atctgcccat cggccctttg gggaggagga atgtgcccaa 300 ggactaaaaa aaggccatgg agccagaggg gcgagggcaa cagacctttc atgggcaaac 360 cttggggccc tgctgtctag catgccccac tacgggtcta ggctgcccat gtaaggaggc 420 aaggcctggg gacacccgag atgcctggtt ataattaacc cagacatgtg gctgcccccc 480 cccccccaac acctgctgcc tctaaaaata accctgtccc tggtggatcc cctgcatgcg 540 aagatcttcg aacaaggctg tgggggactg agggcaggct gtaacaggct tgggggccag 600 ggctttatacg tgcctgggac tcccaaagta ttactgttcc atgttcccgg cgaagggcca 660 gctgtccccc gccagctaga ctcagcactt agtttaggaa ccagtgagca agtcagccct 720 tggggcagcc catacaaggc catggggctg ggcaagctgc acgcctgggt ccggggtggg 780 cacggtgccc gggcaacgag ctgaaagctc atctgctctc aggggcccct ccctggggac 840 agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg gcacaggggc 900 tgccctcatt ctaccaccac ctccacagca cagacagaca ctcaggagca gccagcggcg 960 cgcccaggta agtttagtct ttttgtcttt tatttcaggt cccggatccg gtggtggtgc 1020 aaatcaaaga actgctcctc agtggatgtt gcctttactt ctaggcctgt acggaagtgt 1080 tacttctgct ctaaaagctg cggaattgta cccgcggccg cggctagcca ccatgctgag 1140 ggtcttcatc ctctatgccg agaacgtcca cacacccgac accgacatca gcgatgccta 1200 ctgctccgcg gtgtttgcag gggtgaagaa gagaaccaaa gtcatcaaga acagcgtgaa 1260 ccctgtatgg aatgagggat ttgaatggga cctcaagggc atccccctgg accagggctc 1320 tgagcttcat gtggtggtca aagaccatga gacgatgggg aggaacaggt tcctggggga 1380 agccaaggtc ccactccgag aggtcctcgc cacccctagt ctgtccgcca gcttcaatgc 1440 ccccctgctg gacaccaaga agcagcccac aggggcctcg ctggtcctgc aggtgtccta 1500 cacaccgctg cctggagctg tgcccctgtt cccgccccct actcctctgg agccctcccc 1560 gactctgcct gacctggatg tagtggcaga cacaggagga gaggaagaca cagaggacca 1620 gggactcact ggagatgagg cggagccatt cctggatcaa agcggaggcc cgggggctcc 1680 caccacccca aggaaactac cttcacgtcc tccgccccac taccccggga tcaaaagaaa 1740 gcgaagtgcg cctacatcta gaaagctgct gtcagacaaa ccgcaggatt tccagatcag 1800 ggtccaggtg atcgaggggc gccagctgcc gggggtgaac atcaagcctg tggtcaaggt 1860 taccgctgca gggcagacca agcggacgcg gatccacaag ggaaacagcc cactcttcaa 1920 tgagactctt ttcttcaact tgtttgactc tcctggggag ctgtttgatg agcccatctt 1980 tatcacggtg gtagactctc gttctctcag gacagatgct ctcctcgggg agttccggat 2040 ggacgtgggc accatttaca gagagccccg gcacgcctat ctcaggaagt ggctgctgct 2100 ctcagaccct gatgacttct ctgctggggc cagaggctac ctgaaaacaa gcctttgtgt 2160 gctggggcct ggggacgaag cgcctctgga gagaaaagac ccctctgaag acaaggagga 2220 cattgaaagc aacctgctcc ggcccacagg cgtagccctg cgaggagccc acttctgcct 2280 gaaggtcttc cgggccgagg acttgccgca gatggacgat gccgtgatgg acaacgtgaa 2340 acagatcttt ggcttcgaga gtaacaagaa gaacttggtg gacccctttg tggaggtcag 2400 ctttgcgggg aaaatgctgt gcagcaagat cttggagaag acggccaacc ctcagtgggaa 2460 ccagaacatc acactgcctg ccatgtttcc ctccatgtgc gaaaaaatga ggattcgtat 2520 catagactgg gaccgcctga ctcacaatga catcgtggct accacctacc tgagtatgtc 2580 gaaaatctct gcccctggag gagaaataga agaggagcct gcaggtgctg tcaagccttc 2640 gaaagcctca gacttggatg actacctggg cttcctcccc acttttgggc cctgctacat 2700 caacctctat ggcagtccca gagagttcac aggcttccca gacccctaca cagagctcaa 2760 2820 gctggtggag cacagtgaac agaaggtgga ggaccttcct gcggatgaca tcctccgggt 2880 ggagaagtac cttaggaggc gcaagtactc cctgtttgcg gccttctact cagccaccat 2940 gctgcaggat gtggatgatg ccatccagtt tgaggtcagc atcgggaact acgggaacaa 3000 gttcgacatg acctgcctgc cgctggcctc caccactcag tacagccgtg cagtctttga 3060 cgggtgccac tactactacc taccctgggg taacgtgaaa cctgtggtgg tgctgtcatc 3120 ctactgggag gacatcagcc atagaatcga gactcagaac cagctgcttg ggattgctga 3180 ccggctggaa gctggcctgg agcaggtcca cctggccctg aaggcgcagt gctccacgga 3240 ggacgtggac tcgctggtgg ctcagctgac ggatgagctc atcgcaggct gcagccagcc 3300 tctgggtgac atccatgaga caccctctgc cacccacctg gaccagtacc tgtaccagct 3360 gcgcacccat cacctgagcc aaatcactga ggctgccctg gccctgaagc tcggccacag 3420 tgagctccct gcagctctgg agcaggcgga ggactggctc ctgcgtctgc gtgccctggc 3480 agaggagccc cagaacagcc tgccggacat cgtcatctgg atgctgcagg gagacaagcg 3540 tgtggcatac cagcgggtgc ccgccaccca agtcctcttc tcccggcggg gtgccaacta 3600 ctgtggcaag aattgtggga agctacagac aatctttctg aaatatccga tggagaaggt 3660 gcctggcgcc cggatgccag tgcagatacg ggtcaagctg tggtttgggc tctctgtgga 3720 tgagaaggag ttcaaccagt ttgctgaggg gaagctgtct gtctttgctg aaacctatga 3780 gaacgagact aagttggccc ttgttgggaa ctggggcaca acgggcctca cctaccccaa 3840 gttttctgac gtcacgggca agatcaagct acccaaggac agcttccgcc cctcggccgg 3900 ctggacctgg gctggagatt ggttcgtgtg tccggagaag actctgctcc atgacatgga 3960 cgccggtcac ctgagcttcg tggaagaggt gtttgagaac cagacccggc ttcccggagg 4020 ccagtggatc tacatgagtg acaactacac cgatgtgaac ggggagaagg tgcttcccaa 4080 ggatgacatt gagtgcccac tgggctggaa gtgggaagat gaggaatggt ccacagacct 4140 caaccgggct gtcgatgagc aaggctggga gtatagcatc accatccccc cggagcggaa 4200 gccgaagcac tgggtccctg ctgagaagat gtactacaca caccgacggc ggcgctgggt 4260 gcgcctgcgc aggagggatc tcagccaaat ggaagcactg aaaaggcaca ggcaggcgga 4320 ggcggagggc gagggctggg agtacgcctc tctttttggc tggaagttcc acctcgagta 4380 ccgcaagaca gatgccttcc gccgccgccg ctggcgccgt cgcatggagc cactggagaa 4440 gacggggcct gcagctgtgt ttgcccttga gggcggccgc aataaaagat ctttattttc 4500 attagatctg tgtgttggtt ttttgtgtgt ctagagcatg gcgggttaat cattaactac 4560 aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4620 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 4680 cgagcgcgc 4689 <210> 7 <211> 49 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> PolyA signal <400> 7 aataaaagat ctttattttc attagatctg tgtgttggtt ttttgtgtg 49 <210> 8 <211> 4592 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> DYSF F3' Expression Cassette v1 <400> 8 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctgg 180 tcgactctag agttttcgtc atctggatgc tgcagggaga caagcgtgtg gcataccagc 240 gggtgcccgc ccaccaagtc ctcttctccc ggcggggtgc caactactgt ggcaagaatt 300 gtgggaagct acagacaatc tttctgaaat atccgatgga gaaggtgcct ggcgcccgga 360 tgccagtgca gatacgggtc aagctgtggt ttgggctctc tgtggatgag aaggagttca 420 accagtttgc tgaggggaag ctgtctgtct ttgctgaaac ctatgagaac gagactaagt 480 tggcccttgt tgggaactgg ggcacaacgg gcctcaccta ccccaagttt tctgacgtca 540 cgggcaagat caagctaccc aaggacagct tccgcccctc ggccggctgg acctgggctg 600 gagattggtt cgtgtgtccg gagaagactc tgctccatga catggacgcc ggtcacctga 660 gcttcgtgga agaggtgttt gagaaccaga cccggcttcc cggaggccag tggatctaca 720 tgagtgacaa ctacaccgat gtgaacgggg agaaggtgct tcccaaggat gacattgagt 780 gcccactggg ctggaagtgg gaagatgagg aatggtccac agacctcaac cgggctgtcg 840 atgagcaagg ctgggagtat agcatcacca tccccccgga gcggaagccg aagcactggg 900 tccctgctga gaagatgtac tacacacacc gacggcggcg ctgggtgcgc ctgcgcagga 960 gggatctcag ccaaatggaa gcactgaaaa ggcacaggca ggcggaggcg gagggcgagg 1020 gctgggagta cgcctctctt tttggctgga agttccacct cgagtaccgc aagacagatg 1080 ccttccgccg ccgccgctgg cgccgtcgca tggagccact ggagaagacg gggcctgcag 1140 ctgtgtttgc ccttgagggg gccctgggcg gcgtgatgga tgacaagagt gaagattcca 1200 tgtccgtctc caccttgagc ttcggtgtga acagaccac gatttcctgc atattcgact 1260 atgggaaccg ctaccatcta cgctgctaca tgtaccaggc ccgggacctg gctgcgatgg 1320 acaaggactc tttttctgat ccctatgcca tcgtctcctt cctgcaccag agccagaaga 1380 cggtggtggt gaagaacacc cttaacccca cctgggacca gacgctcatc ttctacgaga 1440 tcgagatctt tggcgagccg gccacagttg ctgagcaacc gcccagcatt gtggtggagc 1500 tgtacgacca tgacacttat ggtgcagacg agtttatggg tcgctgcatc tgtcaaccga 1560 gtctggaacg gatgccacgg ctggcctggt tccccactgac gaggggcagc cagccgtcgg 1620 gggagctgct ggcctctttt gagctcatcc agagagagaa gccggccatc caccatattc 1680 ctggttttga ggtgcaggag acatcaagga tcctggatga gtctgaggac acagacctgc 1740 cctacccacc accccagagg gaggccaaca tctacatggt tcctcagaac atcaagccag 1800 cgctccagcg taccgccatc gagatcctgg catggggcct gcggaacatg aagagttacc 1860 agctggccaa catctcctcc cccagcctcg tggtagagtg tgggggccag acggtgcagt 1920 cctgtgtcat caggaacctc cggaagaacc ccaactttga catctgcacc ctcttcatgg 1980 aagtgatgct gcccagggag gagctctact gcccccccat caccgtcaag gtcatcgata 2040 accgccagtt tggccgccgg cctgtggtgg gccagtgtac catccgctcc ctggagagct 2100 tcctgtgtga cccctactcg gcggagagtc catccccaca gggtggccca gacgatgtga 2160 gcctactcag tcctgggggaa gacgtgctca tcgacattga tgacaaggag cccctcatcc 2220 ccatccagga ggaagagttc atcgattggt ggagcaaatt ctttgcctcc ataggggaga 2280 gggaaaagtg cggctcctac ctggagaagg attttgacac cctgaaggtc tatgacacac 2340 agctggagaa tgtggaggcc tttgagggcc tgtctgactt ttgtaacacc ttcaagctgt 2400 accggggcaa gacgcaggag gagacagaag atccatctgt gattggtgaa tttaagggcc 2460 tcttcaaaat ttatcccctc ccagaagacc cagccatccc catgccccca agacagttcc 2520 accagctggc cgcccaggga ccccaggagt gcttggtccg tatctacatt gtccgagcat 2580 ttggcctgca gcccaaggac cccaatggaa agtgtgatcc ttacatcaag atctccatag 2640 ggaagaaatc agtgagtgac caggataact acatcccctg cacgctggag cccgtatttg 2700 gaaagatgtt cgagctgacc tgcactctgc ctctggagaa ggacctaaag atcactctct 2760 atgactatga cctcctctcc aaggacgaaa agatcggtga gacggtcgtc gacctggaga 2820 acaggctgct gtccaagttt ggggctcgct gtggactccc acagacctac tgtgtctctg 2880 gaccgaacca gtggcgggac cagctccgcc cctcccagct cctccacctc ttctgccagc 2940 agcatagagt caaggcacct gtgtaccgga cagaccgtgt aatgtttcag gataaagaat 3000 attccattga agagatagag gctggcagga tcccaaaccc acacctgggc ccagtggagg 3060 agcgtctggc tctgcatgtg cttcagcagc agggcctggt cccggagcac gtggagtcac 3120 ggcccctcta cagccccctg cagccagaca tcgagcaggg gaagctgcag atgtgggtcg 3180 acctatttcc gaaggccctg gggcggcctg gacctccctt caacatcacc ccacggagag 3240 ccagaaggtt tttcctgcgt tgtattatct ggaataccag agatgtgatc ctggatgacc 3300 tgagcctcac gggggagaag atgagcgaca tttatgtgaa aggttggatg attggctttg 3360 aagaacacaa gcaaaagaca gacgtgcatt atcgttccct gggaggtgaa ggcaacttca 3420 actggaggtt cattttcccc ttcgactacc tgccagctga gcaagtctgt accattgcca 3480 agaaggatgc cttctggagg ctggacaaga ctgagagcaa aatcccagca cgagtggtgt 3540 tccagatctg ggacaatgac aagttctcct ttgatgattt tctgggctcc ctgcagctcg 3600 atctcaaccg catgcccaag ccagccaaga cagccaagaa gtgctccttg gaccagctgg 3660 atgatgcttt ccacccagaa tggtttgtgt ccctttttga gcagaaaaca gtgaagggct 3720 ggtggccctg tgtagcagaa gagggtgaga agaaaatact ggcgggcaag ctggaaatga 3780 ccttggagat tgtagcagag agtgagcatg aggagcggcc tgctggccag ggccgggatg 3840 agcccaacat gaaccctaag cttgaggacc caaggcgccc cgacacctcc ttcctgtggt 3900 ttacctcccc atacaagacc atgaagttca tcctgtggcg gcgtttccgg tgggccatca 3960 tcctcttcat catcctcttc atcctgctgc tgttcctggc catcttcatc tacgccttcc 4020 cgaactatgc tgccatgaag ctggtgaagc ccttcagctg aggactctcc tgccctgtag 4080 aaggggccgt ggggtcccct ccagcatggg actggcctgc ctcctccgcc cagctcggcg 4140 agctcctcca gacctcctag gcctgattgt cctgccaggg tgggcagaca gacagatgga 4200 ccggcccaca ctcccagagt tgctaacatg gagctctgag atcaccccac ttccatcatt 4260 tccttctccc ccaacccaac gcttttttgg atcagctcag acatatttca gtataaaaca 4320 gttggaacca caaaaaaaaa aaaaaaaagt cgacgcggcc gcaataaaag atctttattt 4380 tcattagatc tgtgtgttgg ttttttgtgt gtctagagca tggctacgta gataagtagc 4440 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc 4500 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 4560 cccgggcggc ctcagtgagc gagcgagcgc gc 4592 <210> 9 <211> 1113 <212> PRT <213> Homo sapiens <220> <223> 5' hDYSF protein fragment <400> 9 Met Leu Arg Val Phe Ile Leu Tyr Ala Glu Asn Val His Thr Pro Asp 1 5 10 15 Thr Asp Ile Ser Asp Ala Tyr Cys Ser Ala Val Phe Ala Gly Val Lys 20 25 30 Lys Arg Thr Lys Val Ile Lys Asn Ser Val Asn Pro Val Trp Asn Glu 35 40 45 Gly Phe Glu Trp Asp Leu Lys Gly Ile Pro Leu Asp Gln Gly Ser Glu 50 55 60 Leu His Val Val Val Lys Asp His Glu Thr Met Gly Arg Asn Arg Phe 65 70 75 80 Leu Gly Glu Ala Lys Val Pro Leu Arg Glu Val Leu Ala Thr Pro Ser 85 90 95 Leu Ser Ala Ser Phe Asn Ala Pro Leu Leu Asp Thr Lys Lys Gln Pro 100 105 110 Thr Gly Ala Ser Leu Val Leu Gln Val Ser Tyr Thr Pro Leu Pro Gly 115 120 125 Ala Val Pro Leu Phe Pro Pro Pro Thr Pro Leu Glu Pro Ser Pro Thr 130 135 140 Leu Pro Asp Leu Asp Val Val Ala Asp Thr Gly Gly Glu Glu Asp Thr 145 150 155 160 Glu Asp Gln Gly Leu Thr Gly Asp Glu Ala Glu Pro Phe Leu Asp Gln 165 170 175 Ser Gly Gly Pro Gly Ala Pro Thr Thr Pro Arg Lys Leu Pro Ser Arg 180 185 190 Pro Pro Pro His Tyr Pro Gly Ile Lys Arg Lys Arg Ser Ala Pro Thr 195 200 205 Ser Arg Lys Leu Leu Ser Asp Lys Pro Gln Asp Phe Gln Ile Arg Val 210 215 220 Gln Val Ile Glu Gly Arg Gln Leu Pro Gly Val Asn Ile Lys Pro Val 225 230 235 240 Val Lys Val Thr Ala Ala Gly Gln Thr Lys Arg Thr Arg Ile His Lys 245 250 255 Gly Asn Ser Pro Leu Phe Asn Glu Thr Leu Phe Phe Asn Leu Phe Asp 260 265 270 Ser Pro Gly Glu Leu Phe Asp Glu Pro Ile Phe Ile Thr Val Val Asp 275 280 285 Ser Arg Ser Leu Arg Thr Asp Ala Leu Leu Gly Glu Phe Arg Met Asp 290 295 300 Val Gly Thr Ile Tyr Arg Glu Pro Arg His Ala Tyr Leu Arg Lys Trp 305 310 315 320 Leu Leu Leu Ser Asp Pro Asp Asp Phe Ser Ala Gly Ala Arg Gly Tyr 325 330 335 Leu Lys Thr Ser Leu Cys Val Leu Gly Pro Gly Asp Glu Ala Pro Leu 340 345 350 Glu Arg Lys Asp Pro Ser Glu Asp Lys Glu Asp Ile Glu Ser Asn Leu 355 360 365 Leu Arg Pro Thr Gly Val Ala Leu Arg Gly Ala His Phe Cys Leu Lys 370 375 380 Val Phe Arg Ala Glu Asp Leu Pro Gln Met Asp Asp Ala Val Met Asp 385 390 395 400 Asn Val Lys Gln Ile Phe Gly Phe Glu Ser Asn Lys Lys Asn Leu Val 405 410 415 Asp Pro Phe Val Glu Val Ser Phe Ala Gly Lys Met Leu Cys Ser Lys 420 425 430 Ile Leu Glu Lys Thr Ala Asn Pro Gln Trp Asn Gln Asn Ile Thr Leu 435 440 445 Pro Ala Met Phe Pro Ser Met Cys Glu Lys Met Arg Ile Arg Ile Ile 450 455 460 Asp Trp Asp Arg Leu Thr His Asn Asp Ile Val Ala Thr Thr Tyr Leu 465 470 475 480 Ser Met Ser Lys Ile Ser Ala Pro Gly Gly Glu Ile Glu Glu Glu Pro 485 490 495 Ala Gly Ala Val Lys Pro Ser Lys Ala Ser Asp Leu Asp Asp Tyr Leu 500 505 510 Gly Phe Leu Pro Thr Phe Gly Pro Cys Tyr Ile Asn Leu Tyr Gly Ser 515 520 525 Pro Arg Glu Phe Thr Gly Phe Pro Asp Pro Tyr Thr Glu Leu Asn Thr 530 535 540 Gly Lys Gly Glu Gly Val Ala Tyr Arg Gly Arg Leu Leu Leu Ser Leu 545 550 555 560 Glu Thr Lys Leu Val Glu His Ser Glu Gln Lys Val Glu Asp Leu Pro 565 570 575 Ala Asp Asp Ile Leu Arg Val Glu Lys Tyr Leu Arg Arg Arg Lys Tyr 580 585 590 Ser Leu Phe Ala Ala Phe Tyr Ser Ala Thr Met Leu Gln Asp Val Asp 595 600 605 Asp Ala Ile Gln Phe Glu Val Ser Ile Gly Asn Tyr Gly Asn Lys Phe 610 615 620 Asp Met Thr Cys Leu Pro Leu Ala Ser Thr Thr Gln Tyr Ser Arg Ala 625 630 635 640 Val Phe Asp Gly Cys His Tyr Tyr Tyr Leu Pro Trp Gly Asn Val Lys 645 650 655 Pro Val Val Val Leu Ser Ser Tyr Trp Glu Asp Ile Ser His Arg Ile 660 665 670 Glu Thr Gln Asn Gln Leu Leu Gly Ile Ala Asp Arg Leu Glu Ala Gly 675 680 685 Leu Glu Gln Val His Leu Ala Leu Lys Ala Gln Cys Ser Thr Glu Asp 690 695 700 Val Asp Ser Leu Val Ala Gln Leu Thr Asp Glu Leu Ile Ala Gly Cys 705 710 715 720 Ser Gln Pro Leu Gly Asp Ile His Glu Thr Pro Ser Ala Thr His Leu 725 730 735 Asp Gln Tyr Leu Tyr Gln Leu Arg Thr His His Leu Ser Gln Ile Thr 740 745 750 Glu Ala Ala Leu Ala Leu Lys Leu Gly His Ser Glu Leu Pro Ala Ala 755 760 765 Leu Glu Gln Ala Glu Asp Trp Leu Leu Arg Leu Arg Ala Leu Ala Glu 770 775 780 Glu Pro Gln Asn Ser Leu Pro Asp Ile Val Ile Trp Met Leu Gln Gly 785 790 795 800 Asp Lys Arg Val Ala Tyr Gln Arg Val Pro Ala His Gln Val Leu Phe 805 810 815 Ser Arg Arg Gly Ala Asn Tyr Cys Gly Lys Asn Cys Gly Lys Leu Gln 820 825 830 Thr Ile Phe Leu Lys Tyr Pro Met Glu Lys Val Pro Gly Ala Arg Met 835 840 845 Pro Val Gln Ile Arg Val Lys Leu Trp Phe Gly Leu Ser Val Asp Glu 850 855 860 Lys Glu Phe Asn Gln Phe Ala Glu Gly Lys Leu Ser Val Phe Ala Glu 865 870 875 880 Thr Tyr Glu Asn Glu Thr Lys Leu Ala Leu Val Gly Asn Trp Gly Thr 885 890 895 Thr Gly Leu Thr Tyr Pro Lys Phe Ser Asp Val Thr Gly Lys Ile Lys 900 905 910 Leu Pro Lys Asp Ser Phe Arg Pro Ser Ala Gly Trp Thr Trp Ala Gly 915 920 925 Asp Trp Phe Val Cys Pro Glu Lys Thr Leu Leu His Asp Met Asp Ala 930 935 940 Gly His Leu Ser Phe Val Glu Glu Val Phe Glu Asn Gln Thr Arg Leu 945 950 955 960 Pro Gly Gly Gln Trp Ile Tyr Met Ser Asp Asn Tyr Thr Asp Val Asn 965 970 975 Gly Glu Lys Val Leu Pro Lys Asp Asp Ile Glu Cys Pro Leu Gly Trp 980 985 990 Lys Trp Glu Asp Glu Glu Trp Ser Thr Asp Leu Asn Arg Ala Val Asp 995 1000 1005 Glu Gln Gly Trp Glu Tyr Ser Ile Thr Ile Pro Pro Glu Arg Lys 1010 1015 1020 Pro Lys His Trp Val Pro Ala Glu Lys Met Tyr Tyr Thr His Arg 1025 1030 1035 Arg Arg Arg Trp Val Arg Leu Arg Arg Arg Asp Leu Ser Gln Met 1040 1045 1050 Glu Ala Leu Lys Arg His Arg Gln Ala Glu Ala Glu Gly Glu Gly 1055 1060 1065 Trp Glu Tyr Ala Ser Leu Phe Gly Trp Lys Phe His Leu Glu Tyr 1070 1075 1080 Arg Lys Thr Asp Ala Phe Arg Arg Arg Arg Trp Arg Arg Arg Met 1085 1090 1095 Glu Pro Leu Glu Lys Thr Gly Pro Ala Ala Val Phe Ala Leu Glu 1100 1105 1110 <210> 10 <211> 1287 <212> PRT <213> Homo sapiens <220> <223> 3' hDYSF protein fragment <400> 10 Val Ile Trp Met Leu Gln Gly Asp Lys Arg Val Ala Tyr Gln Arg Val 1 5 10 15 Pro Ala His Gln Val Leu Phe Ser Arg Arg Gly Ala Asn Tyr Cys Gly 20 25 30 Lys Asn Cys Gly Lys Leu Gln Thr Ile Phe Leu Lys Tyr Pro Met Glu 35 40 45 Lys Val Pro Gly Ala Arg Met Pro Val Gln Ile Arg Val Lys Leu Trp 50 55 60 Phe Gly Leu Ser Val Asp Glu Lys Glu Phe Asn Gln Phe Ala Glu Gly 65 70 75 80 Lys Leu Ser Val Phe Ala Glu Thr Tyr Glu Asn Glu Thr Lys Leu Ala 85 90 95 Leu Val Gly Asn Trp Gly Thr Thr Gly Leu Thr Tyr Pro Lys Phe Ser 100 105 110 Asp Val Thr Gly Lys Ile Lys Leu Pro Lys Asp Ser Phe Arg Pro Ser 115 120 125 Ala Gly Trp Thr Trp Ala Gly Asp Trp Phe Val Cys Pro Glu Lys Thr 130 135 140 Leu Leu His Asp Met Asp Ala Gly His Leu Ser Phe Val Glu Glu Val 145 150 155 160 Phe Glu Asn Gln Thr Arg Leu Pro Gly Gly Gln Trp Ile Tyr Met Ser 165 170 175 Asp Asn Tyr Thr Asp Val Asn Gly Glu Lys Val Leu Pro Lys Asp Asp 180 185 190 Ile Glu Cys Pro Leu Gly Trp Lys Trp Glu Asp Glu Glu Trp Ser Thr 195 200 205 Asp Leu Asn Arg Ala Val Asp Glu Gln Gly Trp Glu Tyr Ser Ile Thr 210 215 220 Ile Pro Pro Glu Arg Lys Pro Lys His Trp Val Pro Ala Glu Lys Met 225 230 235 240 Tyr Tyr Thr His Arg Arg Arg Arg Trp Val Arg Leu Arg Arg Arg Asp 245 250 255 Leu Ser Gln Met Glu Ala Leu Lys Arg His Arg Gln Ala Glu Ala Glu 260 265 270 Gly Glu Gly Trp Glu Tyr Ala Ser Leu Phe Gly Trp Lys Phe His Leu 275 280 285 Glu Tyr Arg Lys Thr Asp Ala Phe Arg Arg Arg Arg Trp Arg Arg Arg 290 295 300 Met Glu Pro Leu Glu Lys Thr Gly Pro Ala Ala Val Phe Ala Leu Glu 305 310 315 320 Gly Ala Leu Gly Gly Val Met Asp Asp Lys Ser Glu Asp Ser Met Ser 325 330 335 Val Ser Thr Leu Ser Phe Gly Val Asn Arg Pro Thr Ile Ser Cys Ile 340 345 350 Phe Asp Tyr Gly Asn Arg Tyr His Leu Arg Cys Tyr Met Tyr Gln Ala 355 360 365 Arg Asp Leu Ala Ala Met Asp Lys Asp Ser Phe Ser Asp Pro Tyr Ala 370 375 380 Ile Val Ser Phe Leu His Gln Ser Gln Lys Thr Val Val Val Lys Asn 385 390 395 400 Thr Leu Asn Pro Thr Trp Asp Gln Thr Leu Ile Phe Tyr Glu Ile Glu 405 410 415 Ile Phe Gly Glu Pro Ala Thr Val Ala Glu Gln Pro Pro Ser Ile Val 420 425 430 Val Glu Leu Tyr Asp His Asp Thr Tyr Gly Ala Asp Glu Phe Met Gly 435 440 445 Arg Cys Ile Cys Gln Pro Ser Leu Glu Arg Met Pro Arg Leu Ala Trp 450 455 460 Phe Pro Leu Thr Arg Gly Ser Gln Pro Ser Gly Glu Leu Leu Ala Ser 465 470 475 480 Phe Glu Leu Ile Gln Arg Glu Lys Pro Ala Ile His His Ile Pro Gly 485 490 495 Phe Glu Val Gln Glu Thr Ser Arg Ile Leu Asp Glu Ser Glu Asp Thr 500 505 510 Asp Leu Pro Tyr Pro Pro Pro Gln Arg Glu Ala Asn Ile Tyr Met Val 515 520 525 Pro Gln Asn Ile Lys Pro Ala Leu Gln Arg Thr Ala Ile Glu Ile Leu 530 535 540 Ala Trp Gly Leu Arg Asn Met Lys Ser Tyr Gln Leu Ala Asn Ile Ser 545 550 555 560 Ser Pro Ser Leu Val Val Glu Cys Gly Gly Gln Thr Val Gln Ser Cys 565 570 575 Val Ile Arg Asn Leu Arg Lys Asn Pro Asn Phe Asp Ile Cys Thr Leu 580 585 590 Phe Met Glu Val Met Leu Pro Arg Glu Glu Leu Tyr Cys Pro Pro Ile 595 600 605 Thr Val Lys Val Ile Asp Asn Arg Gln Phe Gly Arg Arg Pro Val Val 610 615 620 Gly Gln Cys Thr Ile Arg Ser Leu Glu Ser Phe Leu Cys Asp Pro Tyr 625 630 635 640 Ser Ala Glu Ser Pro Ser Pro Gln Gly Gly Pro Asp Asp Val Ser Leu 645 650 655 Leu Ser Pro Gly Glu Asp Val Leu Ile Asp Ile Asp Asp Lys Glu Pro 660 665 670 Leu Ile Pro Ile Gln Glu Glu Glu Phe Ile Asp Trp Trp Ser Lys Phe 675 680 685 Phe Ala Ser Ile Gly Glu Arg Glu Lys Cys Gly Ser Tyr Leu Glu Lys 690 695 700 Asp Phe Asp Thr Leu Lys Val Tyr Asp Thr Gln Leu Glu Asn Val Glu 705 710 715 720 Ala Phe Glu Gly Leu Ser Asp Phe Cys Asn Thr Phe Lys Leu Tyr Arg 725 730 735 Gly Lys Thr Gln Glu Glu Thr Glu Asp Pro Ser Val Ile Gly Glu Phe 740 745 750 Lys Gly Leu Phe Lys Ile Tyr Pro Leu Pro Glu Asp Pro Ala Ile Pro 755 760 765 Met Pro Pro Arg Gln Phe His Gln Leu Ala Ala Gln Gly Pro Gln Glu 770 775 780 Cys Leu Val Arg Ile Tyr Ile Val Arg Ala Phe Gly Leu Gln Pro Lys 785 790 795 800 Asp Pro Asn Gly Lys Cys Asp Pro Tyr Ile Lys Ile Ser Ile Gly Lys 805 810 815 Lys Ser Val Ser Asp Gln Asp Asn Tyr Ile Pro Cys Thr Leu Glu Pro 820 825 830 Val Phe Gly Lys Met Phe Glu Leu Thr Cys Thr Leu Pro Leu Glu Lys 835 840 845 Asp Leu Lys Ile Thr Leu Tyr Asp Tyr Asp Leu Leu Ser Lys Asp Glu 850 855 860 Lys Ile Gly Glu Thr Val Val Asp Leu Glu Asn Arg Leu Leu Ser Lys 865 870 875 880 Phe Gly Ala Arg Cys Gly Leu Pro Gln Thr Tyr Cys Val Ser Gly Pro 885 890 895 Asn Gln Trp Arg Asp Gln Leu Arg Pro Ser Gln Leu Leu His Leu Phe 900 905 910 Cys Gln Gln His Arg Val Lys Ala Pro Val Tyr Arg Thr Asp Arg Val 915 920 925 Met Phe Gln Asp Lys Glu Tyr Ser Ile Glu Glu Ile Glu Ala Gly Arg 930 935 940 Ile Pro Asn Pro His Leu Gly Pro Val Glu Glu Arg Leu Ala Leu His 945 950 955 960 Val Leu Gln Gln Gln Gly Leu Val Pro Glu His Val Glu Ser Arg Pro 965 970 975 Leu Tyr Ser Pro Leu Gln Pro Asp Ile Glu Gln Gly Lys Leu Gln Met 980 985 990 Trp Val Asp Leu Phe Pro Lys Ala Leu Gly Arg Pro Gly Pro Pro Phe 995 1000 1005 Asn Ile Thr Pro Arg Arg Ala Arg Arg Phe Phe Leu Arg Cys Ile 1010 1015 1020 Ile Trp Asn Thr Arg Asp Val Ile Leu Asp Asp Leu Ser Leu Thr 1025 1030 1035 Gly Glu Lys Met Ser Asp Ile Tyr Val Lys Gly Trp Met Ile Gly 1040 1045 1050 Phe Glu Glu His Lys Gln Lys Thr Asp Val His Tyr Arg Ser Leu 1055 1060 1065 Gly Gly Glu Gly Asn Phe Asn Trp Arg Phe Ile Phe Pro Phe Asp 1070 1075 1080 Tyr Leu Pro Ala Glu Gln Val Cys Thr Ile Ala Lys Lys Asp Ala 1085 1090 1095 Phe Trp Arg Leu Asp Lys Thr Glu Ser Lys Ile Pro Ala Arg Val 1100 1105 1110 Val Phe Gln Ile Trp Asp Asn Asp Lys Phe Ser Phe Asp Asp Phe 1115 1120 1125 Leu Gly Ser Leu Gln Leu Asp Leu Asn Arg Met Pro Lys Pro Ala 1130 1135 1140 Lys Thr Ala Lys Lys Cys Ser Leu Asp Gln Leu Asp Asp Ala Phe 1145 1150 1155 His Pro Glu Trp Phe Val Ser Leu Phe Glu Gln Lys Thr Val Lys 1160 1165 1170 Gly Trp Trp Pro Cys Val Ala Glu Glu Gly Glu Lys Lys Ile Leu 1175 1180 1185 Ala Gly Lys Leu Glu Met Thr Leu Glu Ile Val Ala Glu Ser Glu 1190 1195 1200 His Glu Glu Arg Pro Ala Gly Gln Gly Arg Asp Glu Pro Asn Met 1205 1210 1215 Asn Pro Lys Leu Glu Asp Pro Arg Arg Pro Asp Thr Ser Phe Leu 1220 1225 1230 Trp Phe Thr Ser Pro Tyr Lys Thr Met Lys Phe Ile Leu Trp Arg 1235 1240 1245 Arg Phe Arg Trp Ala Ile Ile Leu Phe Ile Ile Leu Phe Ile Leu 1250 1255 1260 Leu Leu Phe Leu Ala Ile Phe Ile Tyr Ala Phe Pro Asn Tyr Ala 1265 1270 1275 Ala Met Lys Leu Val Lys Pro Phe Ser 1280 1285 <210> 11 <211> 6914 <212> DNA <213> Homo sapiens <220> <223> hDYSF DNA full length <400> 11 gcggccgccg cccagccagg tgcaaaatgc cgtgtcattg ggagactccg cagccggagc 60 attagattac agctcgacgg agctcgggaa gggcggcggg ggtggaagat gagcagaagc 120 ccctgttctc ggaacgccgg ctgacaagcg gggtgagcgc agccggggcg gggacccagc 180 ctagcccact ggagcagccg ggggtggccc gttccccttt aagagcaact gctctaagcc 240 aggagccaga gattcgagcc ggcctcgccc agccagccct ctccagcgag gggacccaca 300 agcggcgcct cggccctccc gacctttccg agccctcttt gcgccctggg cgcacggggc 360 cctacacgcg ccaagcatgc tgagggtctt catcctctat gccgagaacg tccacacacc 420 cgacaccgac atcagcgatg cctactgctc cgcggtgttt gcaggggtga agaagagaac 480 caaagtcatc aagaacagcg tgaaccctgt atggaatgag ggatttgaat gggacctcaa 540 gggcatcccc ctggaccagg gctctgagct tcatgtggtg gtcaaagacc atgagacgat 600 ggggaggaac aggttcctgg gggaagccaa ggtcccactc cgagaggtcc tcgccacccc 660 tagtctgtcc gccagcttca atgcccccct gctggacacc aagaagcagc ccacaggggc 720 ctcgctggtc ctgcaggtgt cctacacacc gctgcctgga gctgtgcccc tgttcccgcc 780 ccctactcct ctggagccct ccccgactct gcctgacctg gatgtagtgg cagacacagg 840 aggagagggaa gacacagagg accagggact cactggagat gaggcggagc cattcctgga 900 tcaaagcgga ggcccggggg ctcccaccac cccaaggaaa ctaccttcac gtcctccgcc 960 ccactacccc gggatcaaaa gaaagcgaag tgcgcctaca tctagaaagc tgctgtcaga 1020 caaaccgcag gatttccaga tcagggtcca ggtgatcgag gggcgccagc tgccgggggt 1080 gaacatcaag cctgtggtca aggttaccgc tgcagggcag accaagcgga cgcggatcca 1140 caagggaaac agcccactct tcaatgagac tcttttcttc aacttgtttg actctcctgg 1200 ggagctgttt gatgagccca tctttatcac ggtggtagac tctcgttctc tcaggacaga 1260 tgctctcctc ggggagttcc ggatggacgt gggcaccatt tacagagagc cccggcacgc 1320 ctatctcagg aagtggctgc tgctctcaga ccctgatgac ttctctgctg gggccagagg 1380 ctacctgaaa acaagccttt gtgtgctggg gcctggggac gaagcgcctc tggagagaaa 1440 agacccctct gaagacaagg aggacattga aagcaacctg ctccggccca caggcgtagc 1500 cctgcgagga gcccacttct gcctgaaggt cttccgggcc gaggacttgc cgcagatgga 1560 cgatgccgtg atggacaacg tgaaacagat ctttggcttc gagagtaaca agaagaactt 1620 ggtggacccc tttgtggagg tcagctttgc ggggaaaatg ctgtgcagca agatcttgga 1680 gaagacggcc aaccctcagt ggaaccagaa catcacactg cctgccatgt ttccctccat 1740 gtgcgaaaaa atgaggattc gtatcataga ctgggaccgc ctgactcaca atgacatcgt 1800 ggctaccacc tacctgagta tgtcgaaaat ctctgcccct ggaggagaaa tagaagagga 1860 gcctgcaggt gctgtcaagc cttcgaaagc ctcagacttg gatgactacc tgggcttcct 1920 ccccactttt gggccctgct acatcaacct ctatggcagt cccagagagt tcacaggctt 1980 cccagacccc tacacagagc tcaacacagg caagggggaa ggtgtggctt atcgtggccg 2040 gcttctgctc tccctggaga ccaagctggt ggagcacagt gaacagaagg tggaggacct 2100 tcctgcggat gacatcctcc gggtggagaa gtaccttagg aggcgcaagt actccctgtt 2160 tgcggccttc tactcagcca ccatgctgca ggatgtggat gatgccatcc agtttgaggt 2220 cagcatcggg aactacggga acaagttcga catgacctgc ctgccgctgg cctccaccac 2280 tcagtacagc cgtgcagtct ttgacgggtg ccactactac tacctaccct ggggtaacgt 2340 gaaacctgtg gtggtgctgt catcctactg ggaggacatc agccatagaa tcgagactca 2400 gaaccagctg cttgggattg ctgaccggct ggaagctggc ctggagcagg tccacctggc 2460 cctgaaggcg cagtgctcca cggaggacgt ggactcgctg gtggctcagc tgacggatga 2520 gctcatcgca ggctgcagcc agcctctggg tgacatccat gagacaccct ctgccaccca 2580 cctggaccag tacctgtacc agctgcgcac ccatcacctg agccaaatca ctgaggctgc 2640 cctggccctg aagctcggcc acagtgagct ccctgcagct ctggagcagg cggaggactg 2700 gctcctgcgt ctgcgtgccc tggcagagga gccccagaac agcctgccgg acatcgtcat 2760 ctggatgctg cagggagaca agcgtgtggc ataccagcgg gtgcccgccc accaagtcct 2820 cttctcccgg cggggtgcca actactgtgg caagaattgt gggaagctac agacaatctt 2880 tctgaaatat ccgatggaga aggtgcctgg cgcccggatg ccaggtgcaga tacgggtcaa 2940 gctgtggttt gggctctcag tggatgagaa ggagttcaac cagtttgctg aggggaagct 3000 gtctgtcttt gctgaaacct atgagaacga gactaagttg gcccttgttg ggaactgggg 3060 cacaacgggc ctcacctacc ccaagttttc tgacgtcacg ggcaagatca agctacccaa 3120 ggacagcttc cgcccctcgg ccggctggac ctgggctgga gattggttcg tgtgtccgga 3180 gaagactctg ctccatgaca tggacgccgg tcacctgagc ttcgtggaag aggtgtttga 3240 gaaccagacc cggcttcccg gaggccagtg gatctacatg agtgacaact acaccgatgt 3300 gaacggggag aaggtgcttc ccaaggatga cattgagtgc ccactgggct ggaagtggga 3360 agatgagggaa tggtccacag acctcaaccg ggctgtcgat gagcaaggct gggagtatag 3420 catcaccatc cccccggagc ggaagccgaa gcactgggtc cctgctgaga agatgtacta 3480 cacacaccga cggcggcgct gggtgcgcct gcgcaggagg gatctcagcc aaatggaagc 3540 actgaaaagg cacaggcagg cggaggcgga gggcgagggc tgggagtacg cctctctttt 3600 tggctggaag ttccacctcg agtaccgcaa gacagatgcc ttccgccgcc gccgctggcg 3660 ccgtcgcatg gagccactgg agaagacggg gcctgcagct gtgtttgccc ttgagggggc 3720 cctgggcggc gtgatggatg acaagagtga agattccatg tccgtctcca ccttgagctt 3780 cggtgtgaac agacccacga tttcctgcat attcgactat gggaaccgct accatctacg 3840 ctgctacatg taccaggccc gggacctggc tgcgatggac aaggactctt tttctgatcc 3900 ctatgccatc gtctccttcc tgcaccagag ccagaagacg gtggtggtga agaacaccct 3960 taaccccacc tgggaccaga cgctcatctt ctacgagatc gagatctttg gcgagccggc 4020 cacagttgct gagcaaccgc ccagcattgt ggtggagctg tacgaccatg acacttatgg 4080 tgcagacgag tttatgggtc gctgcatctg tcaaccgagt ctggaacgga tgccacggct 4140 ggcctggttc ccactgacga ggggcagcca gccgtcgggg gagctgctgg cctcttttga 4200 gctcatccag agagagaagc cggccatcca ccatattcct ggttttgagg tgcaggagac 4260 atcaaggatc ctggatgagt ctgaggacac agacctgccc tacccaccac cccagaggga 4320 ggccaacatc tacatggttc ctcagaacat caagccagcg ctccagcgta ccgccatcga 4380 gatcctggca tggggcctgc ggaacatgaa gagttaccag ctggccaaca tctcctcccc 4440 cagcctcgtg gtagagtgtg ggggccagac ggtgcagtcc tgtgtcatca ggaacctccg 4500 gaagaacccc aactttgaca tctgcaccct cttcatggaa gtgatgctgc ccagggagga 4560 gctctactgc ccccccatca ccgtcaaggt catcgataac cgccagtttg gccgccggcc 4620 tgtggtgggc cagtgtacca tccgctccct ggagagcttc ctgtgtgacc cctactcggc 4680 ggagagtcca tccccacagg gtggcccaga cgatgtgagc ctactcagtc ctggggaaga 4740 cgtgctcatc gacattgatg acaaggagcc cctcatcccc atccaggagg aagagttcat 4800 cgattggtgg agcaaattct ttgcctccat aggggagagg gaaaagtgcg gctcctacct 4860 ggagaaggat tttgacaccc tgaaggtcta tgacacacag ctggagaatg tggagggcctt 4920 tgagggcctg tctgactttt gtaacacctt caagctgtac cggggcaaga cgcaggagga 4980 gacagaagat ccatctgtga ttggtgaatt taagggcctc ttcaaaattt atcccctccc 5040 agaagaccca gccatcccca tgcccccaag acagttccac cagctggccg cccagggacc 5100 ccaggagtgc ttggtccgta tctacattgt ccgagcattt ggcctgcagc ccaaggaccc 5160 caatggaaag tgtgatcctt acatcaagat ctccataggg aagaaatcag tgagtgacca 5220 ggataactac atcccctgca cgctggagcc cgtatttgga aagatgttcg agctgacctg 5280 cactctgcct ctggagaagg acctaaagat cactctctat gactatgacc tcctctccaa 5340 ggacgaaaag atcggtgaga cggtcgtcga cctggagaac aggctgctgt ccaagtttgg 5400 ggctcgctgt ggactcccac agacctactg tgtctctgga ccgaaccagt ggcgggacca 5460 gctccgcccc tcccagctcc tccacctctt ctgccagcag catagagtca aggcacctgt 5520 gtaccggaca gaccgtgtaa tgtttcagga taaagaatat tccattgaag agatagaggc 5580 tggcaggatc ccaaacccac acctgggccc agtggaggag cgtctggctc tgcatgtgct 5640 tcagcagcag ggcctggtcc cggagcacgt ggagtcacgg cccctctaca gccccctgca 5700 gccagacatc gagcagggga agctgcagat gtgggtcgac ctatttccga aggccctggg 5760 gcggcctgga cctcccttca acatcacccc acggagagcc agaaggtttt tcctgcgttg 5820 tattatctgg aataccagag atgtgatcct ggatgacctg agcctcacgg gggagaagat 5880 gagcgacatt tatgtgaaag gttggatgat tggctttgaa gaacacaagc aaaagacaga 5940 cgtgcattat cgttccctgg gaggtgaagg caacttcaac tggaggttca ttttcccctt 6000 cgactacctg ccagctgagc aagtctgtac cattgccaag aaggatgcct tctggaggct 6060 ggacaagact gagagcaaaa tcccagcacg agtggtgttc cagatctggg acaatgacaa 6120 gttctccttt gatgattttc tgggctccct gcagctcgat ctcaaccgca tgcccaagcc 6180 agccaagaca gccaagaagt gctccttgga ccagctggat gatgctttcc acccagaatg 6240 gtttgtgtcc ctttttgagc agaaaacagt gaagggctgg tggccctgtg tagcagaaga 6300 gggtgagaag aaaatactgg cgggcaagct ggaaatgacc ttggagattg tagcagagag 6360 tgagcatgag gagcggcctg ctggccaggg ccgggatgag cccaacatga accctaagct 6420 tgaggaccca aggcgccccg acacctcctt cctgtggttt acctccccat acaagaccat 6480 gaagttcatc ctgtggcggc gtttccggtg ggccatcatc ctcttcatca tcctcttcat 6540 cctgctgctg ttcctggcca tcttcatcta cgccttcccg aactatgctg ccatgaagct 6600 ggtgaagccc ttcagctgag gactctcctg ccctgtagaa ggggccgtgg ggtcccctcc 6660 agcatgggac tggcctgcct cctccgccca gctcggcgag ctcctccaga cctcctaggc 6720 ctgattgtcc tgccagggtg ggcagacaga cagatggacc ggcccacact cccagagttg 6780 ctaacatgga gctctgagat caccccactt ccatcatttc cttctccccc aacccaacgc 6840 ttttttggat cagctcagac atatttcagt ataaaacagt tggaaccaca aaaaaaaaaa 6900 aaaaaaaaaa aaaa 6914 <210> 12 <211> 2080 <212> PRT <213> Homo sapiens <220> <223> hDYSF protein full length <400> 12 Met Leu Arg Val Phe Ile Leu Tyr Ala Glu Asn Val His Thr Pro Asp 1 5 10 15 Thr Asp Ile Ser Asp Ala Tyr Cys Ser Ala Val Phe Ala Gly Val Lys 20 25 30 Lys Arg Thr Lys Val Ile Lys Asn Ser Val Asn Pro Val Trp Asn Glu 35 40 45 Gly Phe Glu Trp Asp Leu Lys Gly Ile Pro Leu Asp Gln Gly Ser Glu 50 55 60 Leu His Val Val Val Lys Asp His Glu Thr Met Gly Arg Asn Arg Phe 65 70 75 80 Leu Gly Glu Ala Lys Val Pro Leu Arg Glu Val Leu Ala Thr Pro Ser 85 90 95 Leu Ser Ala Ser Phe Asn Ala Pro Leu Leu Asp Thr Lys Lys Gln Pro 100 105 110 Thr Gly Ala Ser Leu Val Leu Gln Val Ser Tyr Thr Pro Leu Pro Gly 115 120 125 Ala Val Pro Leu Phe Pro Pro Pro Thr Pro Leu Glu Pro Ser Pro Thr 130 135 140 Leu Pro Asp Leu Asp Val Val Val Ala Asp Thr Gly Gly Glu Glu Asp Thr 145 150 155 160 Glu Asp Gln Gly Leu Thr Gly Asp Glu Ala Glu Pro Phe Leu Asp Gln 165 170 175 Ser Gly Gly Pro Gly Ala Pro Thr Thr Pro Arg Lys Leu Pro Ser Arg 180 185 190 Pro Pro Pro His Tyr Pro Gly Ile Lys Arg Lys Arg Ser Ala Pro Thr 195 200 205 Ser Arg Lys Leu Leu Ser Asp Lys Pro Gln Asp Phe Gln Ile Arg Val 210 215 220 Gln Val Ile Glu Gly Arg Gln Leu Pro Gly Val Asn Ile Lys Pro Val 225 230 235 240 Val Lys Val Thr Ala Ala Gly Gln Thr Lys Arg Thr Arg Ile His Lys 245 250 255 Gly Asn Ser Pro Leu Phe Asn Glu Thr Leu Phe Phe Asn Leu Phe Asp 260 265 270 Ser Pro Gly Glu Leu Phe Asp Glu Pro Ile Phe Ile Thr Val Val Asp 275 280 285 Ser Arg Ser Leu Arg Thr Asp Ala Leu Leu Gly Glu Phe Arg Met Asp 290 295 300 Val Gly Thr Ile Tyr Arg Glu Pro Arg His Ala Tyr Leu Arg Lys Trp 305 310 315 320 Leu Leu Leu Ser Asp Pro Asp Asp Phe Ser Ala Gly Ala Arg Gly Tyr 325 330 335 Leu Lys Thr Ser Leu Cys Val Leu Gly Pro Gly Asp Glu Ala Pro Leu 340 345 350 Glu Arg Lys Asp Pro Ser Glu Asp Lys Glu Asp Ile Glu Ser Asn Leu 355 360 365 Leu Arg Pro Thr Gly Val Ala Leu Arg Gly Ala His Phe Cys Leu Lys 370 375 380 Val Phe Arg Ala Glu Asp Leu Pro Gln Met Asp Asp Ala Val Met Asp 385 390 395 400 Asn Val Lys Gln Ile Phe Gly Phe Glu Ser Asn Lys Lys Asn Leu Val 405 410 415 Asp Pro Phe Val Glu Val Ser Phe Ala Gly Lys Met Leu Cys Ser Lys 420 425 430 Ile Leu Glu Lys Thr Ala Asn Pro Gln Trp Asn Gln Asn Ile Thr Leu 435 440 445 Pro Ala Met Phe Pro Ser Met Cys Glu Lys Met Arg Ile Arg Ile Ile 450 455 460 Asp Trp Asp Arg Leu Thr His Asn Asp Ile Val Ala Thr Thr Tyr Leu 465 470 475 480 Ser Met Ser Lys Ile Ser Ala Pro Gly Gly Glu Ile Glu Glu Glu Glu Pro 485 490 495 Ala Gly Ala Val Lys Pro Ser Lys Ala Ser Asp Leu Asp Asp Tyr Leu 500 505 510 Gly Phe Leu Pro Thr Phe Gly Pro Cys Tyr Ile Asn Leu Tyr Gly Ser 515 520 525 Pro Arg Glu Phe Thr Gly Phe Pro Asp Pro Tyr Thr Glu Leu Asn Thr 530 535 540 Gly Lys Gly Glu Gly Val Ala Tyr Arg Gly Arg Leu Leu Leu Ser Leu 545 550 555 560 Glu Thr Lys Leu Val Glu His Ser Glu Gln Lys Val Glu Asp Leu Pro 565 570 575 Ala Asp Asp Ile Leu Arg Val Glu Lys Tyr Leu Arg Arg Arg Arg Lys Tyr 580 585 590 Ser Leu Phe Ala Ala Phe Tyr Ser Ala Thr Met Leu Gln Asp Val Asp 595 600 605 Asp Ala Ile Gln Phe Glu Val Ser Ile Gly Asn Tyr Gly Asn Lys Phe 610 615 620 Asp Met Thr Cys Leu Pro Leu Ala Ser Thr Thr Gln Tyr Ser Arg Ala 625 630 635 640 Val Phe Asp Gly Cys His Tyr Tyr Tyr Leu Pro Trp Gly Asn Val Lys 645 650 655 Pro Val Val Val Leu Ser Ser Tyr Trp Glu Asp Ile Ser His Arg Ile 660 665 670 Glu Thr Gln Asn Gln Leu Leu Gly Ile Ala Asp Arg Leu Glu Ala Gly 675 680 685 Leu Glu Gln Val His Leu Ala Leu Lys Ala Gln Cys Ser Thr Glu Asp 690 695 700 Val Asp Ser Leu Val Ala Gln Leu Thr Asp Glu Leu Ile Ala Gly Cys 705 710 715 720 Ser Gln Pro Leu Gly Asp Ile His Glu Thr Pro Ser Ala Thr His Leu 725 730 735 Asp Gln Tyr Leu Tyr Gln Leu Arg Thr His His Leu Ser Gln Ile Thr 740 745 750 Glu Ala Ala Leu Ala Leu Lys Leu Gly His Ser Glu Leu Pro Ala Ala 755 760 765 Leu Glu Gln Ala Glu Asp Trp Leu Leu Arg Leu Arg Ala Leu Ala Glu 770 775 780 Glu Pro Gln Asn Ser Leu Pro Asp Ile Val Ile Trp Met Leu Gln Gly 785 790 795 800 Asp Lys Arg Val Ala Tyr Gln Arg Val Pro Ala His Gln Val Leu Phe 805 810 815 Ser Arg Arg Gly Ala Asn Tyr Cys Gly Lys Asn Cys Gly Lys Leu Gln 820 825 830 Thr Ile Phe Leu Lys Tyr Pro Met Glu Lys Val Pro Gly Ala Arg Met 835 840 845 Pro Val Gln Ile Arg Val Lys Leu Trp Phe Gly Leu Ser Val Asp Glu 850 855 860 Lys Glu Phe Asn Gln Phe Ala Glu Gly Lys Leu Ser Val Phe Ala Glu 865 870 875 880 Thr Tyr Glu Asn Glu Thr Lys Leu Ala Leu Val Gly Asn Trp Gly Thr 885 890 895 Thr Gly Leu Thr Tyr Pro Lys Phe Ser Asp Val Thr Gly Lys Ile Lys 900 905 910 Leu Pro Lys Asp Ser Phe Arg Pro Ser Ala Gly Trp Thr Trp Ala Gly 915 920 925 Asp Trp Phe Val Cys Pro Glu Lys Thr Leu Leu His Asp Met Asp Ala 930 935 940 Gly His Leu Ser Phe Val Glu Glu Val Phe Glu Asn Gln Thr Arg Leu 945 950 955 960 Pro Gly Gly Gln Trp Ile Tyr Met Ser Asp Asn Tyr Thr Asp Val Asn 965 970 975 Gly Glu Lys Val Leu Pro Lys Asp Asp Ile Glu Cys Pro Leu Gly Trp 980 985 990 Lys Trp Glu Asp Glu Glu Trp Ser Thr Asp Leu Asn Arg Ala Val Asp 995 1000 1005 Glu Gln Gly Trp Glu Tyr Ser Ile Thr Ile Pro Pro Glu Arg Lys 1010 1015 1020 Pro Lys His Trp Val Pro Ala Glu Lys Met Tyr Tyr Thr His Arg 1025 1030 1035 Arg Arg Arg Trp Val Arg Leu Arg Arg Arg Asp Leu Ser Gln Met 1040 1045 1050 Glu Ala Leu Lys Arg His Arg Gln Ala G lu Ala Glu Gly Glu Gly 1055 1060 1065 Trp Glu Tyr Ala Ser Leu Phe Gly Trp Lys Phe His Leu Glu Tyr 1070 1075 1080 Arg Lys Thr Asp Ala Phe Arg Arg Arg Arg Trp Arg Arg Arg Met 1085 1090 1095 Glu Pro Leu Glu Lys Thr Gly Pro Ala Ala Val Phe Ala Leu Glu 1100 1105 1110 Gly Ala Leu Gly Gly Val Met Asp Asp Lys Ser Glu Asp Ser Met 1115 1120 1125 Ser Val Ser Thr Leu Ser Phe Gly Val Asn Arg Pro Thr Ile Ser 1130 1135 1140 Cys Ile Phe Asp Tyr Gly Asn Arg Tyr His Leu Arg Cys Tyr Met 1145 1150 1155 Tyr Gln Ala Arg Asp Leu Ala Ala Met Asp Lys Asp Ser Phe Ser 1160 1165 1170 Asp Pro Tyr Ala Ile Val Ser Phe Leu His Gln Ser Gln Lys Thr 1175 1180 1185 Val Val Val Lys Asn Thr Leu Asn Pro Thr Trp Asp Gln Thr Leu 1190 1195 1200 Ile Phe Tyr Glu Ile Glu Ile Phe Gly Glu Pro Ala Thr Val Ala 1205 1210 1215 Glu Gln Pro Pro Ser Ile Val Val Glu Leu Tyr Asp His Asp Thr 1220 1225 1230 Tyr Gly Ala Asp Glu Phe Met Gly Arg Cys Ile Cys Gln Pro Ser 1235 1240 1245 Leu Glu Arg Met Pro Arg Leu Ala Trp Phe Pro Leu Thr Arg Gly 12 50 1255 1260 Ser Gln Pro Ser Gly Glu Leu Leu Ala Ser Phe Glu Leu Ile Gln 1265 1270 1275 Arg Glu Lys Pro Ala Ile His His Ile Pro Gly Phe Glu Val Gln 1280 1285 1290 Glu Thr Ser Arg Ile Leu Asp Glu Ser Glu Asp Thr Asp Leu Pro 1295 1300 1305 Tyr Pro Pro Pro Gln Arg Glu Ala Asn Ile Tyr Met Val Pro Gln 1310 1315 1320 Asn Ile Lys Pro Ala Leu Gln Arg Thr Ala Ile Glu Ile Leu Ala 1325 1330 1335 Trp Gly Leu Arg Asn Met Lys Ser Tyr Gln Leu Ala Asn Ile Ser 1340 1345 1350 Ser Pro Ser Leu Val Val Glu Cys Gly Gly Gln Thr Val Gln Ser 1355 1360 1365 Cys Val Ile Arg Asn Leu Arg Lys Asn Pro Asn Phe Asp Ile Cys 1370 1375 1380 Thr Leu Phe Met Glu Val Met Leu Pro Arg Glu Glu Leu Tyr Cys 1385 1390 1395 Pro Pro Ile Thr Val Lys Val Ile Asp Asn Arg Gln Phe Gly Arg 1400 1405 1410 Arg Pro Val Val Gly Gln Cys Thr Ile Arg Ser Leu Glu Ser Phe 1415 1420 1425 Leu Cys Asp Pro Tyr Ser Ala Glu Ser Pro Ser Pro Gln Gly Gly 1430 1435 1440 Pro Asp Asp Val Ser Leu Leu Ser Pro Gly Glu Asp Val Leu Ile 1445 1450 1455 Asp Ile Asp Asp Lys Glu Pro Leu Ile Pro Ile Gln Glu Glu Glu 1460 1465 1470 Phe Ile Asp Trp Trp Ser Lys Phe Phe Ala Ser Ile Gly Glu Arg 1475 1480 1485 Glu Lys Cys Gly Ser Tyr Leu Glu Lys Asp Phe Asp Thr Leu Lys 1490 1495 1500 Val Tyr Asp Thr Gln Leu Glu Asn Val Glu Ala Phe Glu Gly Leu 1505 1510 1515 Ser Asp Phe Cys Asn Thr Phe Lys Leu Tyr Arg Gly Lys Thr Gln 1520 1525 1530 Glu Glu Thr Glu Asp Pro Ser Val Ile Gly Glu Phe Lys Gly Leu 1535 1540 1545 Phe Lys Ile Tyr Pro Leu Pro Glu Asp Pro Ala Ile Pro Met Pro 1550 1555 1560 Pro Arg Gln Phe His Gln Leu Ala Ala Gln Gly Pro Gln Glu Cys 1565 1570 1575 Leu Val Arg Ile Tyr Ile Val Arg Ala Phe Gly Leu Gln Pro Lys 1580 1585 1590 Asp Pro Asn Gly Lys Cys Asp Pro Tyr Ile Lys Ile Ser Ile Gly 1595 1600 1605 Lys Lys Ser Val Ser Asp Gln Asp Asn Tyr Ile Pro Cys Thr Leu 1610 1615 1620 Glu Pro Val Phe Gly Lys Met Phe Glu Leu Thr Cys Thr Leu Pro 1625 1630 1635 Leu Glu Lys Asp Leu Lys Ile Thr Leu Tyr Asp Tyr Asp Leu Leu 1640 1645 1650 Ser Lys Asp Glu Lys Ile Gly Glu Thr V al Val Asp Leu Glu Asn 1655 1660 1665 Arg Leu Leu Ser Lys Phe Gly Ala Arg Cys Gly Leu Pro Gln Thr 1670 1675 1680 Tyr Cys Val Ser Gly Pro Asn Gln Trp Arg Asp Gln Leu Arg Pro 1685 1690 1695 Ser Gln Leu Leu His Leu Phe Cys Gln Gln His Arg Val Lys Ala 1700 1705 1710 Pro Val Tyr Arg Thr Asp Arg Val Met Phe Gln Asp Lys Glu Tyr 1715 1720 1725 Ser Ile Glu Glu Ile Glu Ala Gly Arg Ile Pro Asn Pro His Leu 1730 1735 1740 Gly Pro Val Glu Glu Arg Leu Ala Leu His Val Leu Gln Gln Gln 1745 1750 1755 Gly Leu Val Pro Glu His Val Glu Ser Arg Pro Leu Tyr Ser Pro 1760 1765 1770 Leu Gln Pro Asp Ile Glu Gln Gly Lys Leu Gln Met Trp Val Asp 1775 1780 1785 Leu Phe Pro Lys Ala Leu Gly Arg Pro Gly Pro Pro Phe Asn Ile 1790 1795 1800 Thr Pro Arg Arg Ala Arg Arg Phe Phe Leu Arg Cys Ile Ile Trp 1805 1810 1815 Asn Thr Arg Asp Val Ile Leu Asp Asp Leu Ser Leu Thr Gly Glu 1820 1825 1830 Lys Met Ser Asp Ile Tyr Val Lys Gly Trp Met Ile Gly Phe Glu 1835 1840 1845 Glu His Lys Gln Lys Thr Asp Val His Tyr Arg Ser Leu Gly Gly 18 50 1855 1860 Glu Gly Asn Phe Asn Trp Arg Phe Ile Phe Pro Phe Asp Tyr Leu 1865 1870 1875 Pro Ala Glu Gln Val Cys Thr Ile Ala Lys Lys Asp Ala Phe Trp 1880 1885 1890 Arg Leu Asp Lys Thr Glu Ser Lys Ile Pro Ala Arg Val Val Phe 1895 1900 1905 Gln Ile Trp Asp Asn Asp Lys Phe Ser Phe Asp Asp Phe Leu Gly 1910 1915 1920 Ser Leu Gln Leu Asp Leu Asn Arg Met Pro Lys Pro Ala Lys Thr 1925 1930 1935 Ala Lys Lys Cys Ser Leu Asp Gln Leu Asp Asp Ala Phe His Pro 1940 1945 1950 Glu Trp Phe Val Ser Leu Phe Glu Gln Lys Thr Val Lys Gly Trp 1955 1960 1965 Trp Pro Cys Val Ala Glu Glu Gly Glu Lys Lys Ile Leu Ala Gly 1970 1975 1980 Lys Leu Glu Met Thr Leu Glu Ile Val Ala Glu Ser Glu His Glu 1985 1990 1995 Glu Arg Pro Ala Gly Gln Gly Arg Asp Glu Pro Asn Met Asn Pro 2000 2005 2010 Lys Leu Glu Asp Pro Arg Arg Pro Asp Thr Ser Phe Leu Trp Phe 2015 2020 2025 Thr Ser Pro Tyr Lys Thr Met Lys Phe Ile Leu Trp Arg Arg Phe 2030 2035 2040 Arg Trp Ala Ile Ile Leu Phe Ile Ile Leu Phe Ile Leu Leu Leu 2045 2050 2055 Phe Leu Ala Ile Phe Ile Tyr Ala Phe Pro Asn Tyr Ala Ala Met 2060 2065 2070Lys Leu Val Lys Pro Phe Ser 2075 2080 <210> 13 <211> 3340 <212> DNA <213> Homo sapiens <220> <223> 5' hDYSF DNA fragment v2 <400> 13 atgctgaggg tcttcatcct ctatgccgag aacgtccaca cacccgacac cgacatcagc 60 gatgcctact gctccgcggt gtttgcaggg gtgaagaaga gaaccaaagt catcaagaac 120 agcgtgaacc ctgtatggaa tgagggattt gaatgggacc tcaagggcat ccccctggac 180 cagggctctg agcttcatgt ggtggtcaaa gaccatgaga cgatggggag gaacaggttc 240 ctgggggaag ccaaggtccc actccgagag gtcctcgcca cccctagtct gtccgccagc 300 ttcaatgccc ccctgctgga caccaagaag cagccacag gggcctcgct ggtcctgcag 360 gtgtcctaca caccgctgcc tggagctgtg cccctgttcc cgccccctac tcctctggag 420 ccctccccga ctctgcctga cctggatgta gtggcagaca caggaggaga ggaagacaca 480 gaggaccagg gactcactgg agatgaggcg gagccattcc tggatcaaag cggaggcccg 540 ggggctccca ccaccccaag gaaactacct tcacgtcctc cgccccacta ccccgggatc 600 aaaagaaagc gaagtgcgcc tacatctaga aagctgctgt cagacaaacc gcaggatttc 660 cagatcaggg tccaggtgat cgaggggcgc cagctgccgg gggtgaacat caagcctggg 720 gtcaaggtta ccgctgcagg gcagaccaag cggacgcgga tccacaaggg aaacagccca 780 ctcttcaatg agactctttt cttcaacttg tttgactctc ctggggagct gtttgatgag 840 cccatcttta tcacggtggt agactctcgt tctctcagga cagatgctct cctcggggag 900 ttccggatgg acgtgggcac catttacaga gagccccggc acgcctatct caggaagtgg 960 ctgctgctct cagaccctga tgacttctct gctggggcca gaggctacct gaaaacaagc 1020 ctttgtgtgc tggggcctgg ggacgaagcg cctctggaga gaaaagaccc ctctgaagac 1080 aaggaggaca ttgaaagcaa cctgctccgg cccacaggcg tagccctgcg aggagcccac 1140 ttctgcctga aggtcttccg ggccgaggac ttgccgcaga tggacgatgc cgtgatggac 1200 aacgtgaaac agatctttgg cttcgagagt aacaagaaga acttggtgga cccctttggg 1260 gaggtcagct ttgcgggggaa aatgctgtgc agcaagatct tggagaagac ggccaaccct 1320 cagtggaacc agaacatcac actgcctgcc atgtttccct ccatgtgcga aaaaatgagg 1380 attcgtatca tagactggga ccgcctgact cacaatgaca tcgtggctac cacctacctg 1440 agtatgtcga aaatctctgc ccctggagga gaaatagaag aggagcctgc aggtgctgtc 1500 aagccttcga aagcctcaga cttggatgac tacctgggct tcctccccac ttttgggccc 1560 tgctacatca acctctatgg cagtcccaga gagttcacag gcttcccaga cccctacaca 1620 gagctcaaca caggcaaggg ggaagggtg gcttatcgtg gccggcttct gctctccctg 1680 gagaccaagc tggtggagca cagtgaacag aaggtggagg accttcctgc ggatgacatc 1740 ctccgggtgg agaagtacct taggaggcgc aagtactccc tgtttgcggc cttctactca 1800 gccaccatgc tgcaggatgt ggatgatgcc atccagtttg aggtcagcat cgggaactac 1860 gggaacaagt tcgacatgac ctgcctgccg ctggcctcca ccactcagta cagccgtgca 1920 gtctttgacg ggtgccacta ctactaccta ccctggggta acgtgaaacc tgtggtggtg 1980 ctgtcatcct actgggagga catcagccat agaatcgaga ctcagaacca gctgcttggg 2040 attgctgacc ggctggaagc tggcctggag caggtccacc tggccctgaa ggcgcagtgc 2100 tccacggagg acgtggactc gctggtggct cagctgacgg atgagctcat cgcaggctgc 2160 agccagcctc tgggtgacat ccatgagaca ccctctgcca cccacctgga ccagtacctg 2220 taccagctgc gcacccatca cctgagccaa atcactgagg ctgccctggc cctgaagctc 2280 ggccacagtg agctccctgc agctctggag caggcggagg actggctcct gcgtctgcgt 2340 gccctggcag aggagcccca gaacagcctg ccggacatcg tcatctggat gctgcaggga 2400 gacaagcgtg tggcatacca gcgggtgccc gcccaccaag tcctcttctc ccggcggggt 2460 gccaactact gtggcaagaa ttgtgggaag ctacagacaa tctttctgaa atatccgatg 2520 gagaaggtgc ctggcgcccg gatgccagtg cagatacggg tcaagctggg gtttgggctc 2580 tcagtggatg agaaggagtt caaccagttt gctgagggga agctgtctgt ctttgctgaa 2640 acctatgaga acgagactaa gttggccctt gttgggaact ggggcacaac gggcctcacc 2700 taccccaagt tttctgacgt cacgggcaag atcaagctac ccaaggacag cttccgcccc 2760 tcggccggct ggacctgggc tggagattgg ttcgtgtgtc cggagaagac tctgctccat 2820 gacatggacg ccggtcacct gagcttcgtg gaagaggtgt ttgagaacca gacccggctt 2880 cccggaggcc agtggatcta catgagtgac aactacaccg atgtgaacgg ggagaaggtg 2940 cttcccaagg atgacattga gtgcccactg ggctggaagt gggaagatga ggaatggtcc 3000 acagacctca accgggctgt cgatgagcaa ggctgggagt atagcatcac catccccccg 3060 gagcggaagc cgaagcactg ggtccctgct gagaagatgt actacacaca ccgacggcgg 3120 cgctgggtgc gcctgcgcag gagggatctc agccaaatgg aagcactgaa aaggcacagg 3180 caggcggagg cggagggcga gggctggggag tacgcctctc tttttggctg gaagttccac 3240 ctcgagtacc gcaagacaga tgccttccgc cgccgccgct ggcgccgtcg catggagcca 3300 ctggagaaga cggggcctgc agctgtgttt gcccttgagg 3340 <210> 14 <211> 3866 <212> DNA <213> Homo sapiens <220> <223> 3' hDYSF DNA fragment v2 <400> 14 tcgtcatctg gatgctgcag ggagacaagc gtgtggcata ccagcgggtg cccgcccacc 60 aagtcctctt ctcccggcgg ggtgccaact actgtggcaa gaattgtggg aagctacaga 120 caatctttct gaaatatccg atggagaagg tgcctggcgc ccggatgcca gtgcagatac 180 gggtcaagct gtggtttggg ctctcagtgg atgagaagga gttcaaccag tttgctgagg 240 ggaagctgtc tgtctttgct gaaacctatg agaacgagac taagttggcc cttgttggga 300 actggggcac aacgggcctc acctacccca agttttctga cgtcacgggc aagatcaagc 360 tacccaagga cagcttccgc ccctcggccg gctggacctg ggctggagat tggttcgtgt 420 gtccggagaa gactctgctc catgacatgg acgccggtca cctgagcttc gtggaagagg 480 tgtttgagaa ccagacccgg cttcccggag gccagtggat ctacatgagt gacaactaca 540 ccgatgtgaa cggggagaag gtgcttccca aggatgacat tgagtgccca ctgggctgga 600 agtgggaaga tgaggaatgg tccacagacc tcaaccgggc tgtcgatgag caaggctggg 660 agtatagcat caccatcccc ccggagcgga agccgaagca ctgggtccct gctgagaaga 720 tgtactacac acaccgacgg cggcgctggg tgcgcctgcg caggagggat ctcagccaaa 780 tggaagcact gaaaaggcac aggcaggcgg aggcggaggg cgagggctgg gagtacgcct 840 ctctttttgg ctggaagttc cacctcgagt accgcaagac agatgccttc cgccgccgcc 900 gctggcgccg tcgcatggag ccactggaga agacggggcc tgcagctgtg tttgcccttg 960 agggggccct gggcggcgtg atggatgaca agagtgaaga ttccatgtcc gtctccacct 1020 tgagcttcgg tgtgaacaga cccacgattt cctgcatatt cgactatggg aaccgctacc 1080 atctacgctg ctacatgtac caggcccggg acctggctgc gatggacaag gactcttttt 1140 ctgatcccta tgccatcgtc tccttcctgc accagagcca gaagacggtg gtggtgaaga 1200 acacccttaa ccccacctgg gaccagacgc tcatcttcta cgagatcgag atctttggcg 1260 agccggccac agttgctgag caaccgccca gcattgtggt ggagctgtac gaccatgaca 1320 cttatggtgc agacgagttt atgggtcgct gcatctgtca accgagtctg gaacggatgc 1380 cacggctggc ctggttccca ctgacgaggg gcagccagcc gtcgggggag ctgctggcct 1440 cttttgagct catccagaga gagaagccgg ccatccacca tattcctggt tttgaggtgc 1500 aggagacatc aaggatcctg gatgagtctg aggacacaga cctgccctac ccaccacccc 1560 agagggaggc cacacatctac atggttcctc agaacatcaa gccagcgctc cagcgtaccg 1620 ccatcgagat cctggcatgg ggcctgcgga acatgaagag ttaccagctg gccaacatct 1680 cctcccccag cctcgtggta gagtgtgggg gccagacggt gcagtcctgt gtcatcagga 1740 acctccggaa gaaccccaac tttgacatct gcaccctctt catggaagtg atgctgccca 1800 gggaggagct ctactgcccc cccatcaccg tcaaggtcat cgataaccgc cagtttggcc 1860 gccggcctgt ggtgggccag tgtaccatcc gctccctgga gagcttcctg tgtgacccct 1920 actcggcgga gagtccatcc ccacagggtg gcccagacga tgtgagccta ctcagtcctg 1980 gggaagacgt gctcatcgac attgatgaca aggagcccct catcccccatc caggaggaag 2040 agttcatcga ttggtggagc aaattctttg cctccatagg ggagagggaa aagtgcggct 2100 cctacctgga gaaggatttt gacaccctga aggtctatga cacacagctg gagaatgtgg 2160 aggcctttga gggcctgtct gacttttgta acaccttcaa gctgtaccgg ggcaagacgc 2220 aggagggagac agaagatcca tctgtgattg gtgaatttaa gggcctcttc aaaatttatc 2280 ccctcccaga agacccagcc atccccatgc ccccaagaca gttccaccag ctggccgccc 2340 agggacccca ggaggtgcttg gtccgtatct acattgtccg agcatttggc ctgcagccca 2400 aggaccccaa tggaaagtgt gatccttaca tcaagatctc catagggaag aaatcagtga 2460 gtgaccagga taactacatc ccctgcacgc tggagcccgt atttggaaag atgttcgagc 2520 tgacctgcac tctgcctctg gagaaggacc taaagatcac tctctatgac tatgacctcc 2580 tctccaagga cgaaaagatc ggtgagacgg tcgtcgacct ggagaacagg ctgctgtcca 2640 agtttggggc tcgctgtgga ctcccacaga cctactgtgt ctctggaccg aaccagtggc 2700 gggaccagct ccgcccctcc cagctcctcc acctcttctg ccagcagcat agagtcaagg 2760 cacctgtgta ccggacagac cgtgtaatgt ttcaggataa agaatattcc attgaagaga 2820 tagaggctgg caggatccca aacccacacc tgggcccagt ggaggagcgt ctggctctgc 2880 atgtgcttca gcagcagggc ctggtcccgg agcacgtgga gtcacggccc ctctacagcc 2940 ccctgcagcc agacatcgag caggggaagc tgcagatgtg ggtcgaccta tttccgaagg 3000 ccctggggcg gcctggacct cccttcaaca tcaccccacg gagagccaga aggtttttcc 3060 tgcgttgtat tatctggaat accagagatg tgatcctgga tgacctgagc ctcacggggg 3120 agaagatgag cgacatttat gtgaaaggtt ggatgattgg ctttgaagaa cacaagcaaa 3180 agacagacgt gcattatcgt tccctgggag gtgaaggcaa cttcaactgg aggttcattt 3240 tccccttcga ctacctgcca gctgagcaag tctgtaccat tgccaagaag gatgccttct 3300 ggaggctgga caagactgag agcaaaatcc cagcacgagt ggtgttccag atctggggaca 3360 atgacaagtt ctcctttgat gattttctgg gctccctgca gctcgatctc aaccgcatgc 3420 ccaagccagc caagacagcc aagaagtgct ccttggacca gctggatgat gctttccacc 3480 cagaatggtt tgtgtccctt tttgagcaga aaacagtgaa gggctggtgg ccctggtgtag 3540 cagaagaggg tgagaagaaa atactggcgg gcaagctgga aatgaccttg gagattgtag 3600 cagagagtga gcatgaggag cggcctgctg gccagggccg ggatgagccc aacatgaacc 3660 ctaagcttga ggacccaagg cgccccgaca cctccttcct gtggtttacc tccccataca 3720 agaccatgaa gttcatcctg tggcggcgtt tccggtgggc catcatcctc ttcatcatcc 3780 tcttcatcct gctgctgttc ctggccatct tcatctacgc cttcccgaac tatgctgcca 3840 tgaagctggt gaagcccttc agctga 3866 <210> 15 <211> 4689 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> DYSF F5' Expresson Cassette v2 <400> 15 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttccttg tagttaatga ttaacccgcc atgctctaga gtttaagctt gcatgtctaa 180 gctagaccct tcagattaaa aataactgag gtaagggcct gggtagggga ggtggtgtga 240 gacgctcctg tctctcctct atctgcccat cggccctttg gggaggagga atgtgcccaa 300 ggactaaaaa aaggccatgg agccagaggg gcgagggcaa cagacctttc atgggcaaac 360 cttggggccc tgctgtctag catgccccac tacgggtcta ggctgcccat gtaaggaggc 420 aaggcctggg gacacccgag atgcctggtt ataattaacc cagacatgtg gctgcccccc 480 cccccccaac acctgctgcc tctaaaaata accctgtccc tggtggatcc cctgcatgcg 540 aagatcttcg aacaaggctg tgggggactg agggcaggct gtaacaggct tgggggccag 600 ggctttatacg tgcctgggac tcccaaagta ttactgttcc atgttcccgg cgaagggcca 660 gctgtccccc gccagctaga ctcagcactt agtttaggaa ccagtgagca agtcagccct 720 tggggcagcc catacaaggc catggggctg ggcaagctgc acgcctgggt ccggggtggg 780 cacggtgccc gggcaacgag ctgaaagctc atctgctctc aggggcccct ccctggggac 840 agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg gcacaggggc 900 tgccctcatt ctaccaccac ctccacagca cagacagaca ctcaggagca gccagcggcg 960 cgcccaggta agtttagtct ttttgtcttt tatttcaggt cccggatccg gtggtggtgc 1020 aaatcaaaga actgctcctc agtggatgtt gcctttactt ctaggcctgt acggaagtgt 1080 tacttctgct ctaaaagctg cggaattgta cccgcggccg cggctagcca ccatgctgag 1140 ggtcttcatc ctctatgccg agaacgtcca cacacccgac accgacatca gcgatgccta 1200 ctgctccgcg gtgtttgcag gggtgaagaa gagaaccaaa gtcatcaaga acagcgtgaa 1260 ccctgtatgg aatgagggat ttgaatggga cctcaagggc atccccctgg accagggctc 1320 tgagcttcat gtggtggtca aagaccatga gacgatgggg aggaacaggt tcctggggga 1380 agccaaggtc ccactccgag aggtcctcgc cacccctagt ctgtccgcca gcttcaatgc 1440 ccccctgctg gacaccaaga agcagcccac aggggcctcg ctggtcctgc aggtgtccta 1500 cacaccgctg cctggagctg tgcccctgtt cccgccccct actcctctgg agccctcccc 1560 gactctgcct gacctggatg tagtggcaga cacaggagga gaggaagaca cagaggacca 1620 gggactcact ggagatgagg cggagccatt cctggatcaa agcggaggcc cgggggctcc 1680 caccacccca aggaaactac cttcacgtcc tccgccccac taccccggga tcaaaagaaa 1740 gcgaagtgcg cctacatcta gaaagctgct gtcagacaaa ccgcaggatt tccagatcag 1800 ggtccaggtg atcgaggggc gccagctgcc gggggtgaac atcaagcctg tggtcaaggt 1860 taccgctgca gggcagacca agcggacgcg gatccacaag ggaaacagcc cactcttcaa 1920 tgagactctt ttcttcaact tgtttgactc tcctggggag ctgtttgatg agcccatctt 1980 tatcacggtg gtagactctc gttctctcag gacagatgct ctcctcgggg agttccggat 2040 ggacgtgggc accatttaca gagagccccg gcacgcctat ctcaggaagt ggctgctgct 2100 ctcagaccct gatgacttct ctgctggggc cagaggctac ctgaaaacaa gcctttgtgt 2160 gctggggcct ggggacgaag cgcctctgga gagaaaagac ccctctgaag acaaggagga 2220 cattgaaagc aacctgctcc ggcccacagg cgtagccctg cgaggagccc acttctgcct 2280 gaaggtcttc cgggccgagg acttgccgca gatggacgat gccgtgatgg acaacgtgaa 2340 acagatcttt ggcttcgaga gtaacaagaa gaacttggtg gacccctttg tggaggtcag 2400 ctttgcgggg aaaatgctgt gcagcaagat cttggagaag acggccaacc ctcagtgggaa 2460 ccagaacatc acactgcctg ccatgtttcc ctccatgtgc gaaaaaatga ggattcgtat 2520 catagactgg gaccgcctga ctcacaatga catcgtggct accacctacc tgagtatgtc 2580 gaaaatctct gcccctggag gagaaataga agaggagcct gcaggtgctg tcaagccttc 2640 gaaagcctca gacttggatg actacctggg cttcctcccc acttttgggc cctgctacat 2700 caacctctat ggcagtccca gagagttcac aggcttccca gacccctaca cagagctcaa 2760 2820 gctggtggag cacagtgaac agaaggtgga ggaccttcct gcggatgaca tcctccgggt 2880 ggagaagtac cttaggaggc gcaagtactc cctgtttgcg gccttctact cagccaccat 2940 gctgcaggat gtggatgatg ccatccagtt tgaggtcagc atcgggaact acgggaacaa 3000 gttcgacatg acctgcctgc cgctggcctc caccactcag tacagccgtg cagtctttga 3060 cgggtgccac tactactacc taccctgggg taacgtgaaa cctgtggtgg tgctgtcatc 3120 ctactgggag gacatcagcc atagaatcga gactcagaac cagctgcttg ggattgctga 3180 ccggctggaa gctggcctgg agcaggtcca cctggccctg aaggcgcagt gctccacgga 3240 ggacgtggac tcgctggtgg ctcagctgac ggatgagctc atcgcaggct gcagccagcc 3300 tctgggtgac atccatgaga caccctctgc cacccacctg gaccagtacc tgtaccagct 3360 gcgcacccat cacctgagcc aaatcactga ggctgccctg gccctgaagc tcggccacag 3420 tgagctccct gcagctctgg agcaggcgga ggactggctc ctgcgtctgc gtgccctggc 3480 agaggagccc cagaacagcc tgccggacat cgtcatctgg atgctgcagg gagacaagcg 3540 tgtggcatac cagcgggtgc ccgccaccca agtcctcttc tcccggcggg gtgccaacta 3600 ctgtggcaag aattgtggga agctacagac aatctttctg aaatatccga tggagaaggt 3660 gcctggcgcc cggatgccag tgcagatacg ggtcaagctg tggtttgggc tctcagtgga 3720 tgagaaggag ttcaaccagt ttgctgaggg gaagctgtct gtctttgctg aaacctatga 3780 gaacgagact aagttggccc ttgttgggaa ctggggcaca acgggcctca cctaccccaa 3840 gttttctgac gtcacgggca agatcaagct acccaaggac agcttccgcc cctcggccgg 3900 ctggacctgg gctggagatt ggttcgtgtg tccggagaag actctgctcc atgacatgga 3960 cgccggtcac ctgagcttcg tggaagaggt gtttgagaac cagacccggc ttcccggagg 4020 ccagtggatc tacatgagtg acaactacac cgatgtgaac ggggagaagg tgcttcccaa 4080 ggatgacatt gagtgcccac tgggctggaa gtgggaagat gaggaatggt ccacagacct 4140 caaccgggct gtcgatgagc aaggctggga gtatagcatc accatccccc cggagcggaa 4200 gccgaagcac tgggtccctg ctgagaagat gtactacaca caccgacggc ggcgctgggt 4260 gcgcctgcgc aggagggatc tcagccaaat ggaagcactg aaaaggcaca ggcaggcgga 4320 ggcggagggc gagggctggg agtacgcctc tctttttggc tggaagttcc acctcgagta 4380 ccgcaagaca gatgccttcc gccgccgccg ctggcgccgt cgcatggagc cactggagaa 4440 gacggggcct gcagctgtgt ttgcccttga gggcggccgc aataaaagat ctttattttc 4500 attagatctg tgtgttggtt ttttgtgtgt ctagagcatg gcgggttaat cattaactac 4560 aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4620 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 4680 cgagcgcgc 4689 <210> 16 <211> 4592 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> DYSF F3' Expression Cassette v2 <400> 16 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctgg 180 tcgactctag agttttcgtc atctggatgc tgcagggaga caagcgtgtg gcataccagc 240 gggtgcccgc ccaccaagtc ctcttctccc ggcggggtgc caactactgt ggcaagaatt 300 gtgggaagct acagacaatc tttctgaaat atccgatgga gaaggtgcct ggcgcccgga 360 tgccagtgca gatacgggtc aagctgtggt ttgggctctc agtggatgag aaggagttca 420 accagtttgc tgaggggaag ctgtctgtct ttgctgaaac ctatgagaac gagactaagt 480 tggcccttgt tgggaactgg ggcacaacgg gcctcaccta ccccaagttt tctgacgtca 540 cgggcaagat caagctaccc aaggacagct tccgcccctc ggccggctgg acctgggctg 600 gagattggtt cgtgtgtccg gagaagactc tgctccatga catggacgcc ggtcacctga 660 gcttcgtgga agaggtgttt gagaaccaga cccggcttcc cggaggccag tggatctaca 720 tgagtgacaa ctacaccgat gtgaacgggg agaaggtgct tcccaaggat gacattgagt 780 gcccactggg ctggaagtgg gaagatgagg aatggtccac agacctcaac cgggctgtcg 840 atgagcaagg ctgggagtat agcatcacca tccccccgga gcggaagccg aagcactggg 900 tccctgctga gaagatgtac tacacacacc gacggcggcg ctgggtgcgc ctgcgcagga 960 gggatctcag ccaaatggaa gcactgaaaa ggcacaggca ggcggaggcg gagggcgagg 1020 gctgggagta cgcctctctt tttggctgga agttccacct cgagtaccgc aagacagatg 1080 ccttccgccg ccgccgctgg cgccgtcgca tggagccact ggagaagacg gggcctgcag 1140 ctgtgtttgc ccttgagggg gccctgggcg gcgtgatgga tgacaagagt gaagattcca 1200 tgtccgtctc caccttgagc ttcggtgtga acagaccac gatttcctgc atattcgact 1260 atgggaaccg ctaccatcta cgctgctaca tgtaccaggc ccgggacctg gctgcgatgg 1320 acaaggactc tttttctgat ccctatgcca tcgtctcctt cctgcaccag agccagaaga 1380 cggtggtggt gaagaacacc cttaacccca cctgggacca gacgctcatc ttctacgaga 1440 tcgagatctt tggcgagccg gccacagttg ctgagcaacc gcccagcatt gtggtggagc 1500 tgtacgacca tgacacttat ggtgcagacg agtttatggg tcgctgcatc tgtcaaccga 1560 gtctggaacg gatgccacgg ctggcctggt tccccactgac gaggggcagc cagccgtcgg 1620 gggagctgct ggcctctttt gagctcatcc agagagagaa gccggccatc caccatattc 1680 ctggttttga ggtgcaggag acatcaagga tcctggatga gtctgaggac acagacctgc 1740 cctacccacc accccagagg gaggccaaca tctacatggt tcctcagaac atcaagccag 1800 cgctccagcg taccgccatc gagatcctgg catggggcct gcggaacatg aagagttacc 1860 agctggccaa catctcctcc cccagcctcg tggtagagtg tgggggccag acggtgcagt 1920 cctgtgtcat caggaacctc cggaagaacc ccaactttga catctgcacc ctcttcatgg 1980 aagtgatgct gcccagggag gagctctact gcccccccat caccgtcaag gtcatcgata 2040 accgccagtt tggccgccgg cctgtggtgg gccagtgtac catccgctcc ctggagagct 2100 tcctgtgtga cccctactcg gcggagagtc catccccaca gggtggccca gacgatgtga 2160 gcctactcag tcctgggggaa gacgtgctca tcgacattga tgacaaggag cccctcatcc 2220 ccatccagga ggaagagttc atcgattggt ggagcaaatt ctttgcctcc ataggggaga 2280 gggaaaagtg cggctcctac ctggagaagg attttgacac cctgaaggtc tatgacacac 2340 agctggagaa tgtggaggcc tttgagggcc tgtctgactt ttgtaacacc ttcaagctgt 2400 accggggcaa gacgcaggag gagacagaag atccatctgt gattggtgaa tttaagggcc 2460 tcttcaaaat ttatcccctc ccagaagacc cagccatccc catgccccca agacagttcc 2520 accagctggc cgcccaggga ccccaggagt gcttggtccg tatctacatt gtccgagcat 2580 ttggcctgca gcccaaggac cccaatggaa agtgtgatcc ttacatcaag atctccatag 2640 ggaagaaatc agtgagtgac caggataact acatcccctg cacgctggag cccgtatttg 2700 gaaagatgtt cgagctgacc tgcactctgc ctctggagaa ggacctaaag atcactctct 2760 atgactatga cctcctctcc aaggacgaaa agatcggtga gacggtcgtc gacctggaga 2820 acaggctgct gtccaagttt ggggctcgct gtggactccc acagacctac tgtgtctctg 2880 gaccgaacca gtggcgggac cagctccgcc cctcccagct cctccacctc ttctgccagc 2940 agcatagagt caaggcacct gtgtaccgga cagaccgtgt aatgtttcag gataaagaat 3000 attccattga agagatagag gctggcagga tcccaaaccc acacctgggc ccagtggagg 3060 agcgtctggc tctgcatgtg cttcagcagc agggcctggt cccggagcac gtggagtcac 3120 ggcccctcta cagccccctg cagccagaca tcgagcaggg gaagctgcag atgtgggtcg 3180 acctatttcc gaaggccctg gggcggcctg gacctccctt caacatcacc ccacggagag 3240 ccagaaggtt tttcctgcgt tgtattatct ggaataccag agatgtgatc ctggatgacc 3300 tgagcctcac gggggagaag atgagcgaca tttatgtgaa aggttggatg attggctttg 3360 aagaacacaa gcaaaagaca gacgtgcatt atcgttccct gggaggtgaa ggcaacttca 3420 actggaggtt cattttcccc ttcgactacc tgccagctga gcaagtctgt accattgcca 3480 agaaggatgc cttctggagg ctggacaaga ctgagagcaa aatcccagca cgagtggtgt 3540 tccagatctg ggacaatgac aagttctcct ttgatgattt tctgggctcc ctgcagctcg 3600 atctcaaccg catgcccaag ccagccaaga cagccaagaa gtgctccttg gaccagctgg 3660 atgatgcttt ccacccagaa tggtttgtgt ccctttttga gcagaaaaca gtgaagggct 3720 ggtggccctg tgtagcagaa gagggtgaga agaaaatact ggcgggcaag ctggaaatga 3780 ccttggagat tgtagcagag agtgagcatg aggagcggcc tgctggccag ggccgggatg 3840 agcccaacat gaaccctaag cttgaggacc caaggcgccc cgacacctcc ttcctgtggt 3900 ttacctcccc atacaagacc atgaagttca tcctgtggcg gcgtttccgg tgggccatca 3960 tcctcttcat catcctcttc atcctgctgc tgttcctggc catcttcatc tacgccttcc 4020 cgaactatgc tgccatgaag ctggtgaagc ccttcagctg aggactctcc tgccctgtag 4080 aaggggccgt ggggtcccct ccagcatggg actggcctgc ctcctccgcc cagctcggcg 4140 agctcctcca gacctcctag gcctgattgt cctgccaggg tgggcagaca gacagatgga 4200 ccggcccaca ctcccagagt tgctaacatg gagctctgag atcaccccac ttccatcatt 4260 tccttctccc ccaacccaac gcttttttgg atcagctcag acatatttca gtataaaaca 4320 gttggaacca caaaaaaaaa aaaaaaaagt cgacgcggcc gcaataaaag atctttattt 4380 tcattagatc tgtgtgttgg ttttttgtgt gtctagagca tggctacgta gataagtagc 4440 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc 4500 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 4560 cccgggcggc ctcagtgagc gagcgagcgc gc 4592 <210> 17 <211> 128 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> 3' ITR <400> 17 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120 gagcgcgc 128 <210> 18 <211> 8341 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> Plasmid sequence for DYS F5 Expression Cassette <400> 18 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttccttg tagttaatga ttaacccgcc atgctctaga gtttaagctt gcatgtctaa 180 gctagaccct tcagattaaa aataactgag gtaagggcct gggtagggga ggtggtgtga 240 gacgctcctg tctctcctct atctgcccat cggccctttg gggaggagga atgtgcccaa 300 ggactaaaaa aaggccatgg agccagaggg gcgagggcaa cagacctttc atgggcaaac 360 cttggggccc tgctgtctag catgccccac tacgggtcta ggctgcccat gtaaggaggc 420 aaggcctggg gacacccgag atgcctggtt ataattaacc cagacatgtg gctgcccccc 480 cccccccaac acctgctgcc tctaaaaata accctgtccc tggtggatcc cctgcatgcg 540 aagatcttcg aacaaggctg tgggggactg agggcaggct gtaacaggct tgggggccag 600 ggctttatacg tgcctgggac tcccaaagta ttactgttcc atgttcccgg cgaagggcca 660 gctgtccccc gccagctaga ctcagcactt agtttaggaa ccagtgagca agtcagccct 720 tggggcagcc catacaaggc catggggctg ggcaagctgc acgcctgggt ccggggtggg 780 cacggtgccc gggcaacgag ctgaaagctc atctgctctc aggggcccct ccctggggac 840 agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg gcacaggggc 900 tgccctcatt ctaccaccac ctccacagca cagacagaca ctcaggagca gccagcggcg 960 cgcccaggta agtttagtct ttttgtcttt tatttcaggt cccggatccg gtggtggtgc 1020 aaatcaaaga actgctcctc agtggatgtt gcctttactt ctaggcctgt acggaagtgt 1080 tacttctgct ctaaaagctg cggaattgta cccgcggccg cggctagcca ccatgctgag 1140 ggtcttcatc ctctatgccg agaacgtcca cacacccgac accgacatca gcgatgccta 1200 ctgctccgcg gtgtttgcag gggtgaagaa gagaaccaaa gtcatcaaga acagcgtgaa 1260 ccctgtatgg aatgagggat ttgaatggga cctcaagggc atccccctgg accagggctc 1320 tgagcttcat gtggtggtca aagaccatga gacgatgggg aggaacaggt tcctggggga 1380 agccaaggtc ccactccgag aggtcctcgc cacccctagt ctgtccgcca gcttcaatgc 1440 ccccctgctg gacaccaaga agcagcccac aggggcctcg ctggtcctgc aggtgtccta 1500 cacaccgctg cctggagctg tgcccctgtt cccgccccct actcctctgg agccctcccc 1560 gactctgcct gacctggatg tagtggcaga cacaggagga gaggaagaca cagaggacca 1620 gggactcact ggagatgagg cggagccatt cctggatcaa agcggaggcc cgggggctcc 1680 caccacccca aggaaactac cttcacgtcc tccgccccac taccccggga tcaaaagaaa 1740 gcgaagtgcg cctacatcta gaaagctgct gtcagacaaa ccgcaggatt tccagatcag 1800 ggtccaggtg atcgaggggc gccagctgcc gggggtgaac atcaagcctg tggtcaaggt 1860 taccgctgca gggcagacca agcggacgcg gatccacaag ggaaacagcc cactcttcaa 1920 tgagactctt ttcttcaact tgtttgactc tcctggggag ctgtttgatg agcccatctt 1980 tatcacggtg gtagactctc gttctctcag gacagatgct ctcctcgggg agttccggat 2040 ggacgtgggc accatttaca gagagccccg gcacgcctat ctcaggaagt ggctgctgct 2100 ctcagaccct gatgacttct ctgctggggc cagaggctac ctgaaaacaa gcctttgtgt 2160 gctggggcct ggggacgaag cgcctctgga gagaaaagac ccctctgaag acaaggagga 2220 cattgaaagc aacctgctcc ggcccacagg cgtagccctg cgaggagccc acttctgcct 2280 gaaggtcttc cgggccgagg acttgccgca gatggacgat gccgtgatgg acaacgtgaa 2340 acagatcttt ggcttcgaga gtaacaagaa gaacttggtg gacccctttg tggaggtcag 2400 ctttgcgggg aaaatgctgt gcagcaagat cttggagaag acggccaacc ctcagtgggaa 2460 ccagaacatc acactgcctg ccatgtttcc ctccatgtgc gaaaaaatga ggattcgtat 2520 catagactgg gaccgcctga ctcacaatga catcgtggct accacctacc tgagtatgtc 2580 gaaaatctct gcccctggag gagaaataga agaggagcct gcaggtgctg tcaagccttc 2640 gaaagcctca gacttggatg actacctggg cttcctcccc acttttgggc cctgctacat 2700 caacctctat ggcagtccca gagagttcac aggcttccca gacccctaca cagagctcaa 2760 2820 gctggtggag cacagtgaac agaaggtgga ggaccttcct gcggatgaca tcctccgggt 2880 ggagaagtac cttaggaggc gcaagtactc cctgtttgcg gccttctact cagccaccat 2940 gctgcaggat gtggatgatg ccatccagtt tgaggtcagc atcgggaact acgggaacaa 3000 gttcgacatg acctgcctgc cgctggcctc caccactcag tacagccgtg cagtctttga 3060 cgggtgccac tactactacc taccctgggg taacgtgaaa cctgtggtgg tgctgtcatc 3120 ctactgggag gacatcagcc atagaatcga gactcagaac cagctgcttg ggattgctga 3180 ccggctggaa gctggcctgg agcaggtcca cctggccctg aaggcgcagt gctccacgga 3240 ggacgtggac tcgctggtgg ctcagctgac ggatgagctc atcgcaggct gcagccagcc 3300 tctgggtgac atccatgaga caccctctgc cacccacctg gaccagtacc tgtaccagct 3360 gcgcacccat cacctgagcc aaatcactga ggctgccctg gccctgaagc tcggccacag 3420 tgagctccct gcagctctgg agcaggcgga ggactggctc ctgcgtctgc gtgccctggc 3480 agaggagccc cagaacagcc tgccggacat cgtcatctgg atgctgcagg gagacaagcg 3540 tgtggcatac cagcgggtgc ccgccaccca agtcctcttc tcccggcggg gtgccaacta 3600 ctgtggcaag aattgtggga agctacagac aatctttctg aaatatccga tggagaaggt 3660 gcctggcgcc cggatgccag tgcagatacg ggtcaagctg tggtttgggc tctctgtgga 3720 tgagaaggag ttcaaccagt ttgctgaggg gaagctgtct gtctttgctg aaacctatga 3780 gaacgagact aagttggccc ttgttgggaa ctggggcaca acgggcctca cctaccccaa 3840 gttttctgac gtcacgggca agatcaagct acccaaggac agcttccgcc cctcggccgg 3900 ctggacctgg gctggagatt ggttcgtgtg tccggagaag actctgctcc atgacatgga 3960 cgccggtcac ctgagcttcg tggaagaggt gtttgagaac cagacccggc ttcccggagg 4020 ccagtggatc tacatgagtg acaactacac cgatgtgaac ggggagaagg tgcttcccaa 4080 ggatgacatt gagtgcccac tgggctggaa gtgggaagat gaggaatggt ccacagacct 4140 caaccgggct gtcgatgagc aaggctggga gtatagcatc accatccccc cggagcggaa 4200 gccgaagcac tgggtccctg ctgagaagat gtactacaca caccgacggc ggcgctgggt 4260 gcgcctgcgc aggagggatc tcagccaaat ggaagcactg aaaaggcaca ggcaggcgga 4320 ggcggagggc gagggctggg agtacgcctc tctttttggc tggaagttcc acctcgagta 4380 ccgcaagaca gatgccttcc gccgccgccg ctggcgccgt cgcatggagc cactggagaa 4440 gacggggcct gcagctgtgt ttgcccttga gggcggccgc aataaaagat ctttattttc 4500 attagatctg tgtgttggtt ttttgtgtgt ctagagcatg gcgggttaat cattaactac 4560 aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4620 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 4680 cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4740 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4800 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4860 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4920 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 4980 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 5040 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 5100 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 5160 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5220 ccggtaacta tcgtcttgag tccaacccgg taagacacga ctttcgcca ctggcagcag 5280 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5340 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 5400 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5460 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5520 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5580 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 5640 gttttaaatc aatctaaagt atatatgagt aaaaatattc cggaattgcc agctggggcg 5700 ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg 5760 atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc gtttcgcatg 5820 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 5880 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 5940 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 6000 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 6060 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 6120 ctcctgtcat cccaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 6180 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 6240 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 6300 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 6360 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 6420 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 6480 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 6540 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 6600 gagttcttct gaaccggtaa tattattgaa gcatttatca gggttattgt ctcatgagcg 6660 gatacatatt tgaatgtatt tagaaaata aacaaatagg ggttccgcgc acatttcccc 6720 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 6780 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac 6840 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 6900 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat 6960 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa 7020 ggagaaaata ccgcatcagg cgattccaac atccaataaa tcatacaggc aaggcaaaga 7080 attagcaaaa ttaagcaata aagcctcaga gcataaagct aaatcggttg taccaaaaac 7140 attatgaccc tgtaatactt ttgcgggaga agcctttatt tcaacgcaag gataaaaatt 7200 tttagaaccc tcatatattt taaatgcaat gcctgagtaa tgtgtaggta aagattcaaa 7260 cgggtgagaa aggccggaga cagtcaaatc accatcaata tgatattcaa ccgttctagc 7320 tgataaattc atgccggaga gggtagctat ttttgagagg tctctacaaa ggctatcagg 7380 tcattgcctg agagtctgga gcaaacaaga gaatcgatga acggtaatcg taaaactagc 7440 atgtcaatca tatgtacccc ggttgataat cagaaaagcc ccaaaaacag gaagatgta 7500 taagcaaata tttaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt 7560 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 7620 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 7680 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 7740 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 7800 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 7860 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 7920 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgta ctatggttgc 7980 tttgacgagc acgtataacg tgctttcctc gttagaatca gagcgggagc taaacaggag 8040 gccgattaaa gggattttag acaggaacgg tacgccagaa tcctgagaag tgtttttata 8100 atcagtgagg ccaccgagta aaagagtctg tccatcacgc aaattaaccg ttgtcgcaat 8160 acttctttga ttagtaataa catcacttgc ctgagtagaa gaactcaaac tatcggcctt 8220 gctggtaata tccagaacaa tattaccgcc agccattgca acggaatcgc cattcgccat 8280 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 8340 t8341 <210> 19 <211> 8244 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> Plasmid sequence for DYS F3 Expression Cassette <400> 19 gtagccatgc tctggtcgac tctagagttt tcgtcatctg gatgctgcag ggagacaagc 60 gtgtggcata ccagcgggtg cccgcccacc aagtcctctt ctcccggcgg ggtgccaact 120 actgtggcaa gaattgtggg aagctacaga caatctttct gaaatatccg atggagaagg 180 tgcctggcgc ccggatgcca gtgcagatac gggtcaagct gtggtttggg ctctctgtgg 240 atgagaagga gttcaaccag tttgctgagg ggaagctgtc tgtctttgct gaaacctatg 300 agaacgagac taagttggcc cttgttggga actggggcac aacgggcctc acctacccca 360 agtttctga cgtcacgggc aagatcaagc tacccaagga cagcttccgc ccctcggccg 420 gctggacctg ggctggagat tggttcgtgt gtccggagaa gactctgctc catgacatgg 480 acgccggtca cctgagcttc gtggaagagg tgtttgagaa ccagacccgg cttcccggag 540 gccagtggat ctacatgagt gacaactaca ccgatgtgaa cggggagaag gtgcttccca 600 aggatgacat tgagtgccca ctgggctgga agtgggaaga tgaggaatgg tccacagacc 660 tcaaccgggc tgtcgatgag caaggctggg agtatagcat caccatcccc ccggagcgga 720 agccgaagca ctgggtccct gctgagaaga tgtactacac acaccgacgg cggcgctggg 780 tgcgcctgcg caggagggat ctcagccaaa tggaagcact gaaaaggcac aggcaggcgg 840 aggcggaggg cgagggctgg gagtacgcct ctctttttgg ctggaagttc cacctcgagt 900 accgcaagac agatgccttc cgccgccgcc gctggcgccg tcgcatggag ccactggaga 960 agacggggcc tgcagctgtg tttgcccttg agggggccct gggcggcgtg atggatgaca 1020 agagtgaaga ttccatgtcc gtctccacct tgagcttcgg tgtgaacaga cccacgattt 1080 cctgcatatt cgactatggg aaccgctacc atctacgctg ctacatgtac caggcccggg 1140 acctggctgc gatggacaag gactcttttt ctgatcccta tgccatcgtc tccttcctgc 1200 accagagcca gaagacggtg gtggtgaaga acacccttaa ccccacctgg gaccagacgc 1260 tcatcttcta cgagatcgag atctttggcg agccggccac agttgctgag caaccgccca 1320 gcattgtggt ggagctgtac gaccatgaca cttatggtgc agacgagttt atgggtcgct 1380 gcatctgtca accgagtctg gaacggatgc cacggctggc ctggttccca ctgacgaggg 1440 gcagccagcc gtcgggggag ctgctggcct cttttgagct catccagaga gagaagccgg 1500 ccatccacca tattcctggt tttgaggtgc aggagacatc aaggatcctg gatgagtctg 1560 aggacacaga cctgccctac ccaccacccc agagggaggc caacatctac atggttcctc 1620 agaacatcaa gccagcgctc cagcgtaccg ccatcgagat cctggcatgg ggcctgcgga 1680 acatgaagag ttaccagctg gccaacatct cctcccccag cctcgtggta gagtgtgggg 1740 gccagacggt gcagtcctgt gtcatcagga acctccggaa gaaccccaac tttgacatct 1800 gcaccctctt catggaagtg atgctgccca gggaggagct ctactgcccc cccatcaccg 1860 tcaaggtcat cgataaccgc cagtttggcc gccggcctgt ggtgggccag tgtaccatcc 1920 gctccctgga gagcttcctg tgtgacccct actcggcgga gagtccatcc ccacagggtg 1980 gcccagacga tgtgagccta ctcagtcctg gggaagacgt gctcatcgac attgatgaca 2040 aggagcccct catccccatc caggaggaag agttcatcga ttggtggagc aaattctttg 2100 cctccatagg ggagagggaa aagtgcggct cctacctgga gaaggatttt gacaccctga 2160 aggtctatga cacacagctg gagaatgtgg aggcctttga gggcctgtct gacttttgta 2220 acaccttcaa gctgtaccgg ggcaagacgc aggagggagac agaagatcca tctgtgattg 2280 gtgaatttaa gggcctcttc aaaatttatc ccctcccaga agacccagcc atccccatgc 2340 ccccaagaca gttccaccag ctggccgccc agggacccca ggagtgcttg gtccgtatct 2400 acattgtccg agcatttggc ctgcagccca aggaccccaa tggaaagtgt gatccttaca 2460 tcaagatctc catagggaag aaatcagtga gtgaccagga taactacatc ccctgcacgc 2520 tggagcccgt atttggaaag atgttcgagc tgacctgcac tctgcctctg gagaaggacc 2580 taaagatcac tctctatgac tatgacctcc tctccaagga cgaaaagatc ggtgagacgg 2640 tcgtcgacct ggagaacagg ctgctgtcca agtttggggc tcgctgtgga ctcccacaga 2700 cctactgtgt ctctggaccg aaccagtggc gggaccagct ccgcccctcc cagctcctcc 2760 acctcttctg ccagcagcat agagtcaagg cacctgtgta ccggacagac cgtgtaatgt 2820 ttcaggataa agaatattcc attgaagaga tagaggctgg caggatccca aacccacacc 2880 tgggcccagt ggaggagcgt ctggctctgc atgtgcttca gcagcagggc ctggtcccgg 2940 agcacgtgga gtcacggccc ctctacagcc ccctgcagcc agacatcgag caggggaagc 3000 tgcagatgtg ggtcgaccta tttccgaagg ccctggggcg gcctggacct cccttcaaca 3060 tcaccccacg gagagccaga aggtttttcc tgcgttgtat tatctggaat accagagatg 3120 tgatcctgga tgacctgagc ctcacggggg agaagatgag cgacatttat gtgaaaggtt 3180 ggatgattgg ctttgaagaa cacaagcaaa agacagacgt gcattatcgt tccctggggag 3240 gtgaaggcaa cttcaactgg aggttcattt tccccttcga ctacctgcca gctgagcaag 3300 tctgtaccat tgccaagaag gatgccttct ggaggctgga caagactgag agcaaaatcc 3360 cagcacgagt ggtgttccag atctggggaca atgacaagtt ctcctttgat gattttctgg 3420 gctccctgca gctcgatctc aaccgcatgc ccaagccagc caagacagcc aagaagtgct 3480 ccttggacca gctggatgat gctttccacc cagaatggtt tgtgtccctt tttgagcaga 3540 aaacagtgaa gggctggtgg ccctgtgtag cagaagaggg tgagaagaaa atactggcgg 3600 gcaagctgga aatgaccttg gagattgtag cagagagtga gcatgaggag cggcctgctg 3660 gccagggccg ggatgagccc aacatgaacc ctaagcttga ggacccaagg cgccccgaca 3720 cctccttcct gtggtttacc tccccataca agaccatgaa gttcatcctg tggcggcgtt 3780 tccggtgggc catcatcctc ttcatcatcc tcttcatcct gctgctgttc ctggccatct 3840 tcatctacgc cttcccgaac tatgctgcca tgaagctggt gaagcccttc agctgaggac 3900 tctcctgccc tgtagaaggg gccgtggggt cccctccagc atgggactgg cctgcctcct 3960 ccgcccagct cggcgagctc ctccagacct cctaggcctg attgtcctgc cagggtggggc 4020 agacagacag atggaccggc ccacactccc agagttgcta acatggagct ctgagatcac 4080 cccacttcca tcatttcctt ctcccccaac ccaacgcttt tttggatcag ctcagacata 4140 tttcagtata aaacagttgg aaccacaaaa aaaaaaaaaa aaagtcgacg cggccgcaat 4200 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgtcta gagcatggct 4260 acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt 4320 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc 4380 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg 4440 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 4500 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4560 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 4620 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 4680 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 4740 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 4800 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 4860 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 4920 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 4980 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 5040 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 5100 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 5160 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 5220 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 5280 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 5340 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 5400 atatgagtaa aaatattccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc 5460 cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa 5520 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 5580 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 5640 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 5700 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 5760 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 5820 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatcc caccttgctc 5880 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 5940 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 6000 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 6060 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 6120 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 6180 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 6240 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 6300 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga accggtaata 6360 ttatattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 6420 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta 6480 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 6540 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 6600 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 6660 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt 6720 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 6780 attccaacat ccaataaatc atacaggcaa ggcaaagaat tagcaaaatt aagcaataaa 6840 gcctcagagc ataaagctaa atcggttgta ccaaaaacat tatgaccctg taatactttt 6900 gcgggagaag cctttatttc aacgcaagga taaaaatttt tagaaccctc atatatttta 6960 aatgcaatgc ctgagtaatg tgtaggtaaa gattcaaacg ggtgagaaag gccggagaca 7020 gtcaaatcac catcaatatg atattcaacc gttctagctg ataaattcat gccggagagg 7080 gtagctattt ttgagaggtc tctacaaagg ctatcaggtc attgcctgag agtctggagc 7140 aaacaagaga atcgatgaac ggtaatcgta aaactagcat gtcaatcata tgtaccccgg 7200 ttgataatca gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa 7260 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc 7320 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga 7380 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag 7440 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt 7500 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta 7560 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag 7620 cgggcgctag ggcgctggca aggttagcgg tcacgctgcg cgtaaccacc acacccgccg 7680 cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgagcac gtataacgtg 7740 ctttcctcgt tagaatcaga gcgggagcta aacaggaggc cgattaaagg gattttagac 7800 aggaacggta cgccagaatc ctgagaagtg tttttataat cagtgaggcc accgagtaaa 7860 agagtctgtc catcacgcaa attaaccgtt gtcgcaatac ttctttgatt agtaataaca 7920 tcacttgcct gagtagaaga actcaaacta tcggccttgc tggtaatatc cagaacaata 7980 ttaccgccag ccattgcaac ggaatcgcca ttcgccattc aggctgcgca actgttggga 8040 agggcgatcg gtgcgggcct cttcgctatt acgccagctg cgcgctcgct cgctcactga 8100 ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga 8160 gcgagcgcgc agagagggag tggccaactc catcactagg ggttccttgt agttaatgat 8220 taacccgcca tgctacttat ctac 8244 <210> 20 <211> 7906 <212> DNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <223> full DYF expression cassette <400> 20 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120 gggttccttg tagttaatga ttaacccgcc atgctctaga gtttaagctt gcatgtctaa 180 gctagaccct tcagattaaa aataactgag gtaagggcct gggtagggga ggtggtgtga 240 gacgctcctg tctctcctct atctgcccat cggccctttg gggaggagga atgtgcccaa 300 ggactaaaaa aaggccatgg agccagaggg gcgagggcaa cagacctttc atgggcaaac 360 cttggggccc tgctgtctag catgccccac tacgggtcta ggctgcccat gtaaggaggc 420 aaggcctggg gacacccgag atgcctggtt ataattaacc cagacatgtg gctgcccccc 480 cccccccaac acctgctgcc tctaaaaata accctgtccc tggtggatcc cctgcatgcg 540 aagatcttcg aacaaggctg tgggggactg agggcaggct gtaacaggct tgggggccag 600 ggctttatacg tgcctgggac tcccaaagta ttactgttcc atgttcccgg cgaagggcca 660 gctgtccccc gccagctaga ctcagcactt agtttaggaa ccagtgagca agtcagccct 720 tggggcagcc catacaaggc catggggctg ggcaagctgc acgcctgggt ccggggtggg 780 cacggtgccc gggcaacgag ctgaaagctc atctgctctc aggggcccct ccctggggac 840 agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg gcacaggggc 900 tgccctcatt ctaccaccac ctccacagca cagacagaca ctcaggagca gccagcggcg 960 cgcccaggta agtttagtct ttttgtcttt tatttcaggt cccggatccg gtggtggtgc 1020 aaatcaaaga actgctcctc agtggatgtt gcctttactt ctaggcctgt acggaagtgt 1080 tacttctgct ctaaaagctg cggaattgta cccgcggccg cggctagcca ccatgctgag 1140 ggtcttcatc ctctatgccg agaacgtcca cacacccgac accgacatca gcgatgccta 1200 ctgctccgcg gtgtttgcag gggtgaagaa gagaaccaaa gtcatcaaga acagcgtgaa 1260 ccctgtatgg aatgagggat ttgaatggga cctcaagggc atccccctgg accagggctc 1320 tgagcttcat gtggtggtca aagaccatga gacgatgggg aggaacaggt tcctggggga 1380 agccaaggtc ccactccgag aggtcctcgc cacccctagt ctgtccgcca gcttcaatgc 1440 ccccctgctg gacaccaaga agcagcccac aggggcctcg ctggtcctgc aggtgtccta 1500 cacaccgctg cctggagctg tgcccctgtt cccgccccct actcctctgg agccctcccc 1560 gactctgcct gacctggatg tagtggcaga cacaggagga gaggaagaca cagaggacca 1620 gggactcact ggagatgagg cggagccatt cctggatcaa agcggaggcc cgggggctcc 1680 caccacccca aggaaactac cttcacgtcc tccgccccac taccccggga tcaaaagaaa 1740 gcgaagtgcg cctacatcta gaaagctgct gtcagacaaa ccgcaggatt tccagatcag 1800 ggtccaggtg atcgaggggc gccagctgcc gggggtgaac atcaagcctg tggtcaaggt 1860 taccgctgca gggcagacca agcggacgcg gatccacaag ggaaacagcc cactcttcaa 1920 tgagactctt ttcttcaact tgtttgactc tcctggggag ctgtttgatg agcccatctt 1980 tatcacggtg gtagactctc gttctctcag gacagatgct ctcctcgggg agttccggat 2040 ggacgtgggc accatttaca gagagccccg gcacgcctat ctcaggaagt ggctgctgct 2100 ctcagaccct gatgacttct ctgctggggc cagaggctac ctgaaaacaa gcctttgtgt 2160 gctggggcct ggggacgaag cgcctctgga gagaaaagac ccctctgaag acaaggagga 2220 cattgaaagc aacctgctcc ggcccacagg cgtagccctg cgaggagccc acttctgcct 2280 gaaggtcttc cgggccgagg acttgccgca gatggacgat gccgtgatgg acaacgtgaa 2340 acagatcttt ggcttcgaga gtaacaagaa gaacttggtg gacccctttg tggaggtcag 2400 ctttgcgggg aaaatgctgt gcagcaagat cttggagaag acggccaacc ctcagtgggaa 2460 ccagaacatc acactgcctg ccatgtttcc ctccatgtgc gaaaaaatga ggattcgtat 2520 catagactgg gaccgcctga ctcacaatga catcgtggct accacctacc tgagtatgtc 2580 gaaaatctct gcccctggag gagaaataga agaggagcct gcaggtgctg tcaagccttc 2640 gaaagcctca gacttggatg actacctggg cttcctcccc acttttgggc cctgctacat 2700 caacctctat ggcagtccca gagagttcac aggcttccca gacccctaca cagagctcaa 2760 2820 gctggtggag cacagtgaac agaaggtgga ggaccttcct gcggatgaca tcctccgggt 2880 ggagaagtac cttaggaggc gcaagtactc cctgtttgcg gccttctact cagccaccat 2940 gctgcaggat gtggatgatg ccatccagtt tgaggtcagc atcgggaact acgggaacaa 3000 gttcgacatg acctgcctgc cgctggcctc caccactcag tacagccgtg cagtctttga 3060 cgggtgccac tactactacc taccctgggg taacgtgaaa cctgtggtgg tgctgtcatc 3120 ctactgggag gacatcagcc atagaatcga gactcagaac cagctgcttg ggattgctga 3180 ccggctggaa gctggcctgg agcaggtcca cctggccctg aaggcgcagt gctccacgga 3240 ggacgtggac tcgctggtgg ctcagctgac ggatgagctc atcgcaggct gcagccagcc 3300 tctgggtgac atccatgaga caccctctgc cacccacctg gaccagtacc tgtaccagct 3360 gcgcacccat cacctgagcc aaatcactga ggctgccctg gccctgaagc tcggccacag 3420 tgagctccct gcagctctgg agcaggcgga ggactggctc ctgcgtctgc gtgccctggc 3480 agaggagccc cagaacagcc tgccggacat cgtcatctgg atgctgcagg gagacaagcg 3540 tgtggcatac cagcgggtgc ccgccaccca agtcctcttc tcccggcggg gtgccaacta 3600 ctgtggcaag aattgtggga agctacagac aatctttctg aaatatccga tggagaaggt 3660 gcctggcgcc cggatgccag tgcagatacg ggtcaagctg tggtttgggc tctctgtgga 3720 tgagaaggag ttcaaccagt ttgctgaggg gaagctgtct gtctttgctg aaacctatga 3780 gaacgagact aagttggccc ttgttgggaa ctggggcaca acgggcctca cctaccccaa 3840 gttttctgac gtcacgggca agatcaagct acccaaggac agcttccgcc cctcggccgg 3900 ctggacctgg gctggagatt ggttcgtgtg tccggagaag actctgctcc atgacatgga 3960 cgccggtcac ctgagcttcg tggaagaggt gtttgagaac cagacccggc ttcccggagg 4020 ccagtggatc tacatgagtg acaactacac cgatgtgaac ggggagaagg tgcttcccaa 4080 ggatgacatt gagtgcccac tgggctggaa gtgggaagat gaggaatggt ccacagacct 4140 caaccgggct gtcgatgagc aaggctggga gtatagcatc accatccccc cggagcggaa 4200 gccgaagcac tgggtccctg ctgagaagat gtactacaca caccgacggc ggcgctgggt 4260 gcgcctgcgc aggagggatc tcagccaaat ggaagcactg aaaaggcaca ggcaggcgga 4320 ggcggagggc gagggctggg agtacgcctc tctttttggc tggaagttcc acctcgagta 4380 ccgcaagaca gatgccttcc gccgccgccg ctggcgccgt cgcatggagc cactggagaa 4440 gacggggcct gcagctgtgt ttgcccttga gggggccctg ggcggcgtga tggatgacaa 4500 gagtgaagat tccatgtccg tctccacctt gagcttcggt gtgaacagac ccacgatttc 4560 ctgcatattc gactatggga accgctacca tctacgctgc tacatgtacc aggcccggga 4620 cctggctgcg atggacaagg actctttttc tgatccctat gccatcgtct ccttcctgca 4680 ccagagccag aagacggtgg tggtgaagaa cacccttaac cccacctggg accagacgct 4740 catcttctac gagatcgaga tctttggcga gccggccaca gttgctgagc aaccgcccag 4800 cattgtggtg gagctgtacg accatgacac ttatggtgca gacgagttta tgggtcgctg 4860 catctgtcaa ccgagtctgg aacggatgcc acggctggcc tggttcccac tgacgagggg 4920 cagccagccg tcgggggagc tgctggcctc ttttgagctc atccagagag agaagccggc 4980 catccaccat attcctggtt ttgaggtgca ggagacatca aggatcctgg atgagtctga 5040 ggacacagac ctgccctacc caccacccca gagggaggcc aacatctaca tggttcctca 5100 gaacatcaag ccagcgctcc agcgtaccgc catcgagatc ctggcatggg gcctgcggaa 5160 catgaagagt taccagctgg ccaacatctc ctcccccagc ctcgtggtag agtgtggggg 5220 ccagacggtg cagtcctgtg tcatcaggaa cctccggaag aaccccaact ttgacatctg 5280 caccctcttc atggaagtga tgctgcccag ggaggagctc tactgccccc ccatcaccgt 5340 caaggtcatc gataaccgcc agtttggccg ccggcctgtg gtgggccagt gtaccatccg 5400 ctccctggag agcttcctgt gtgaccccta ctcggcggag agtccatccc cacagggtgg 5460 cccagacgat gtgagcctac tcagtcctgg ggaagacgtg ctcatcgaca ttgatgacaa 5520 ggagcccctc atccccatcc aggaggaaga gttcatcgat tggtggagca aattctttgc 5580 ctccataggg gagagggaaa agtgcggctc ctacctggag aaggattttg acaccctgaa 5640 ggtctatgac acacagctgg agaatgtgga ggcctttgag ggcctgtctg acttttgtaa 5700 caccttcaag ctgtaccggg gcaagacgca ggaggagaca gaagatccat ctgtgattgg 5760 tgaatttaag ggcctcttca aaatttatcc cctcccagaa gacccagcca tccccatgcc 5820 cccaagacag ttccaccagc tggccgccca gggaccccag gagtgcttgg tccgtatcta 5880 cattgtccga gcatttggcc tgcagcccaa ggaccccaat ggaaagtgtg atccttacat 5940 caagatctcc atagggaaga aatcagtgag tgaccaggat aactacatcc cctgcacgct 6000 ggagcccgta tttgggaaaga tgttcgagct gacctgcact ctgcctctgg agaaggacct 6060 aaagatcact ctctatgact atgacctcct ctccaaggac gaaaagatcg gtgagacggt 6120 cgtcgacctg gagaacaggc tgctgtccaa gtttggggct cgctgtggac tcccacagac 6180 ctactgtgtc tctggaccga accagtggcg ggaccagctc cgcccctccc agctcctcca 6240 cctcttctgc cagcagcata gagtcaaggc acctgtgtac cggacagacc gtgtaatgtt 6300 tcaggataaa gaatattcca ttgaagagat agaggctggc aggatcccaa acccacacct 6360 gggcccagtg gaggagcgtc tggctctgca tgtgcttcag cagcagggcc tggtcccgga 6420 gcacgtggag tcacggcccc tctacagccc cctgcagcca gacatcgagc aggggaagct 6480 gcagatgtgg gtcgacctat ttccgaaggc cctggggcgg cctggacctc ccttcaacat 6540 caccccacgg agagccagaa ggtttttcct gcgttgtatt atctggaata ccagagatgt 6600 gatcctggat gacctgagcc tcacggggga gaagatgagc gacatttatg tgaaaggttg 6660 gatgattggc tttgaagaac acaagcaaaa gacagacgtg cattatcgtt ccctgggagg 6720 tgaaggcaac ttcaactgga ggttcatttt ccccttcgac tacctgccag ctgagcaagt 6780 ctgtaccatt gccaagaagg atgccttctg gaggctggac aagactgaga gcaaaatccc 6840 agcacgagtg gtgttccaga tctggggacaa tgacaagttc tcctttgatg attttctggg 6900 ctccctgcag ctcgatctca accgcatgcc caagccagcc aagacagcca agaagtgctc 6960 cttggaccag ctggatgatg ctttccaccc agaatggttt gtgtcccttt ttgagcagaa 7020 aacagtgaag ggctggtggc cctggttagc agaagagggt gagaagaaaa tactggcggg 7080 caagctgggaa atgaccttgg agattgtagc agagagtgag catgaggagc ggcctgctgg 7140 ccagggccgg gatgagccca acatgaaccc taagcttgag gacccaaggc gccccgacac 7200 ctccttcctg tggtttacct ccccatacaa gaccatgaag ttcatcctgt ggcggcgttt 7260 ccggtgggcc atcatcctct tcatcatcct cttcatcctg ctgctgttcc tggccatctt 7320 catctacgcc ttcccgaact atgctgccat gaagctggtg aagcccttca gctgaggact 7380 ctcctgccct gtagaagggg ccgtggggtc ccctccagca tgggactggc ctgcctcctc 7440 cgcccagctc ggcgagctcc tccagacctc ctaggcctga ttgtcctgcc agggtgggca 7500 gacagacaga tggaccggcc cacactccca gagttgctaa catggagctc tgagatcacc 7560 ccacttccat catttccttc tcccccaacc caacgctttt ttggatcagc tcagacatat 7620 ttcagtataa aacagttgga accacaaaaa aaaaaaaaaa aagtcgacgc ggccgcaata 7680 aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtgtctag agcatggcta 7740 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt 7800 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 7860 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgc 7906 <210> 21 <211> 6 <212> PRT <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 21 His His His His His His 1 5 <210> 22 <211> 250 <212> RNA <213> artificial sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(250) <223> This sequence may encompass 100-250 nucleotides <400> 22 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 120 180 240 aaaaaaaaaa 250

Claims (135)

A recombinant polynucleotide encoding a fragment of human dysferlin (hDYSF) protein,
The recombinant polynucleotide comprises a first nucleotide sequence, wherein the first nucleotide sequence is:
(a) the nucleotide sequence of SEQ ID NO: 1, 6, or 18;
(b) at least 90%, 91%, 92% of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 18 over their respective full length of SEQ ID NO: 1, 6, or 18; 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences;
(c) the nucleotide sequence of SEQ ID NO: 13 or 15;
(d) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 13 or 15 over their respective full length of SEQ ID NO: 13 or 15; , 98%, or 99% identical nucleotide sequences;
(e) a nucleotide sequence encoding a fragment of the hDYSF protein, the fragment consisting of the amino acid sequence of SEQ ID NO: 9; or
(f) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleotide sequence of (e) over the entire length of the nucleotide sequence of (e); %, or 99% identical nucleotide sequences;
Consisting of, a recombinant polynucleotide. According to claim 1,
Further comprising one or more additional nucleotide sequences selected from an inverted terminal repeat (ITR), promoter, intron, selection marker, or origin of replication (ORI) To, recombinant polynucleotide. According to claim 2,
wherein the ITR is an AAV ITR. According to claim 3,
wherein the AAV ITR is an AAV2 ITR or an AAV3 ITR. According to any one of claims 2 to 4,
Wherein the recombinant polynucleotide comprises two ITRs. According to any one of claims 2 to 5,
Wherein the ITR comprises the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 17. According to any one of claims 2 to 6,
The promoter is a recombinant polynucleotide comprising a muscle-specific promoter. According to claim 7,
The muscle-specific promoter includes human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, myocyte-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid a-myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK7) promoter, C5-12 promoter, murine creatine kinase enhancer element , skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factor ( A recombinant polynucleotide selected from hypoxia-inducible nuclear factor). According to claim 7,
Wherein the muscle-specific promoter comprises the MHCK7 promoter. According to any one of claims 2 to 6,
The promoter comprises a recombinant promoter, recombinant polynucleotide. According to claim 10,
Wherein the recombinant promoter comprises a recombinant muscle-specific promoter. According to any one of claims 2 to 11,
The promoter comprises the nucleotide sequence of SEQ ID NO: 4, recombinant polynucleotide. According to any one of claims 2 to 12,
The recombinant polynucleotide of claim 1, wherein the intron comprises a 5' donor site, a branch point, and/or a 3' splice site. According to any one of claims 2 to 13,
The recombinant polynucleotide, wherein the intron includes a chimeric intron. According to any one of claims 2 to 14,
The recombinant polynucleotide of claim 1, wherein the intron comprises a 5' donor site from a human β-globin gene. According to any one of claims 2 to 15,
The intron comprises a branch point from an immunoglobulin G (IgG) heavy chain. According to any one of claims 2 to 16,
The recombinant polynucleotide of claim 1, wherein the intron comprises a 3' splice acceptor site from an immunoglobulin G (IgG) heavy chain. According to any one of claims 2 to 17,
The intron comprises the nucleotide sequence of SEQ ID NO: 5, recombinant polynucleotide. According to any one of claims 2 to 18,
The selection marker is an antibiotic resistance gene, recombinant polynucleotide. According to claim 19,
The antibiotic resistance gene is a β-lactamase gene or kanamycin resistance gene, recombinant polynucleotide. According to claim 1,
The recombinant polynucleotide comprises a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 18. According to any one of claims 1 to 21,
Wherein the recombinant nucleotide further does not include a second polynucleotide sequence encoding a second fragment of the hDYSF protein. 23. The method of any one of claims 1 to 22,
wherein the recombinant nucleotide does not contain AAV sequences other than one or more ITRs. 23. The method of any one of claims 1 to 22,
wherein the recombinant nucleotide does not contain viral sequences other than one or more ITRs. A recombinant polynucleotide sequence encoding a fragment of human dysferrin protein,
The recombinant polynucleotide comprises a second nucleotide sequence, wherein the second nucleotide sequence:
(a) the nucleotide sequence of SEQ ID NO: 2, 8 or 19;
(b) at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the nucleotide sequence of SEQ ID NO: 2, 8 or 19 over their entire length of SEQ ID NO: 2, 8 or 19; , 97%, 98%, or 99% identical nucleotide sequences;
(c) the nucleotide sequence of SEQ ID NO: 14 or 16;
(d) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence of SEQ ID NO: 14 or 16 over their respective full length of SEQ ID NO: 14 or 16; 98%, or 99% identical nucleotide sequences;
(e) a polynucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or
(f) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the polynucleotide sequence of (e) above over the entire length of the nucleotide sequence of (d) above %, or 99% identical polynucleotide sequences;
A recombinant polynucleotide sequence consisting of. 26. The method of claim 25,
Further comprising one or more additional nucleotides comprising an inverted terminal repeat (ITR), selectable marker, origin of replication (ORI), untranslated region (UTR), or polyadenylation (polyA) signal , Recombinant Polynucleotide. 27. The method of claim 26,
wherein the ITR is an AAV ITR. 28. The method of claim 27,
wherein the AAV ITR is an AAV2 ITR or an AAV3 ITR. The method of any one of claims 25, 27 and 28,
Wherein the recombinant nucleotide further comprises two ITRs. According to any one of claims 26 to 29,
The ITR comprises a nucleotide sequence of SEQ ID NO: 3 or 17, recombinant polynucleotide. According to any one of claims 26 to 30,
The polyA signal is an artificial polyA signal, recombinant polynucleotide. According to any one of claims 26 to 31,
The polyA signal comprises the nucleotide sequence of SEQ ID NO: 7, recombinant polynucleotide. According to any one of claims 26 to 32,
The recombinant polynucleotide of claim 1, wherein the selectable marker comprises an antibiotic resistance gene. 34. The method of claim 33,
The antibiotic resistance gene comprises a β-lactamase gene or a kanamycin resistance gene. 26. The method of claim 25,
The recombinant polynucleotide comprises a nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 19. According to any one of claims 25 to 35,
Wherein the recombinant nucleotide further does not include a first polynucleotide sequence encoding a first fragment of the hDYSF protein. According to any one of claims 25 to 36,
wherein the recombinant nucleotide does not contain AAV sequences other than one or more ITRs. According to any one of claims 25 to 36,
wherein the recombinant nucleotide does not contain viral sequences other than one or more ITRs. A dual adeno-associated viral (AAV) vector system comprising:
(a) a first AAV vector, wherein the first AAV vector comprises the recombinant polynucleotide of any one of claims 1-24; and
(b) a second AAV vector, wherein the second AAV vector comprises the recombinant polynucleotide of any one of claims 25-38;
A double adeno-associated virus (AAV) vector system comprising a. 25. An adeno-associated virus (AAV) vector comprising the recombinant polynucleotide of any one of claims 1-24. 41. The method of claim 40,
The AAV vectors are AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV- 12, AAV-13, AAVrh.10, AAVrh.20, or AAVrh.74. 41. The method of claim 40,
wherein the AAV vector is AAVrh.74. 39. An adeno-associated virus (AAV) vector comprising the recombinant polynucleotide of any one of claims 25-38. 44. The method of claim 43,
The AAV vectors are AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV- 12, AAV-13, AAVrh.10, AAVrh.20, or AAVrh.74. 44. The method of claim 43,
wherein the AAV vector is AAVrh.74. A composition comprising the recombinant polynucleotide of any one of claims 1 - 38 or the AAV vector of any one of claims 40 - 45 . As a composition,
(a) a first recombinant adeno-associated viral (rAAV) vector, wherein the first rAAV vector comprises the AAV vector of any one of claims 40-42; and
(b) a second rAAV vector, wherein the second rAAV vector comprises the AAV vector of any one of claims 43-45;
A composition comprising a. 48. The method of claim 47,
wherein the molar ratio of the first and second rAAV vectors is about 100:1-1:100, about 10:1-1:10, about 2:1-1:2, or about 1:1. As an adeno-associated virus (AAV) vector, the AAV vector:
(a) a first inverted terminal repeat (ITR);
(b) a polynucleotide encoding a fragment of a human dysferlin (hDYSF) protein, wherein the polynucleotide comprises:
i. the nucleotide sequence of SEQ ID NO: 1 or 6;
ii. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of SEQ ID NO: 1 or 6 over the entire length of SEQ ID NO: 1 or 6 identical nucleotide sequences;
iii. the nucleotide sequence of SEQ ID NO: 13 or 15;
iv. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of SEQ ID NO: 13 or 15 over the entire length of SEQ ID NO: 13 or 15 identical nucleotide sequences;
v. A nucleotide sequence encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 9; or
vi. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of (v) over the entire length of the nucleotide sequence of (v) % identical nucleotide sequence;
made up of; and
(c) a second ITR;
Including,
wherein the polynucleotide is flanked by the first and second ITRs. 50. The method of claim 49,
An AAV vector, further comprising one or more additional polynucleotides selected from a promoter, intron, selectable marker, or origin of replication (ORI). 51. The method of claim 49 or 50,
wherein the ITR is an AAV ITR. 51. The method of claim 51,
wherein the AAV ITR is an AAV2 ITR or an AAV3 ITR. The method of any one of claims 49 to 52,
wherein the first and/or second ITR comprises the nucleotide sequence of SEQ ID NO: 3 or 17. The method of any one of claims 50 to 53,
AAV vector, wherein the promoter is a muscle-specific promoter. 55. The method of claim 54,
The muscle-specific promoter is a myosin heavy chain complex-E box muscle creatine kinase fusion enhancer/promoter, AAV vector. The method of any one of claims 50 to 53,
The promoter is a recombinant promoter, AAV vector. 57. The method of claim 56,
The AAV vector, wherein the recombinant promoter is a recombinant muscle-specific promoter. 58. The method of claim 57,
AAV vector, wherein the recombinant muscle-specific promoter is the MHCK7 promoter. 59. The method of any one of claims 50 to 58,
AAV vector, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 4. 60. The method of any one of claims 50 to 59,
wherein the intron comprises a 5' donor site, a branch point, and/or a 3' splice site. According to any one of claims 50 to 60,
The AAV vector, wherein the intron is a chimeric intron. According to any one of claims 50 to 61,
wherein the intron comprises a 5' donor site from the human β-globin gene. 63. The method of any one of claims 50 to 62,
wherein the intron comprises a branch point from an immunoglobulin G (IgG) heavy chain. The method of any one of claims 50 to 63,
wherein the intron comprises a 3' splice acceptor site from an immunoglobulin G (IgG) heavy chain. The method of any one of claims 50 to 64,
The AAV vector, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 5. 66. The method of any one of claims 50 to 65,
AAV vector, wherein the selectable marker is an antibiotic resistance gene. 67. The method of claim 66,
The antibiotic resistance gene is a β-lactamase gene or a kanamycin resistance gene, AAV vector. The method of any one of claims 49 to 67,
The AAV vector comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 15. 69. The method of any one of claims 49 to 68,
Wherein the AAV vector does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein. According to any one of claims 49 to 69,
wherein the AAV vector does not contain AAV sequences other than one or more ITRs. According to any one of claims 49 to 69,
wherein the AAV vector does not contain viral sequences other than one or more ITRs. As an adeno-associated virus (AAV) vector, the AAV vector:
(a) a first inverted terminal repeat (ITR);
(b) a polynucleotide encoding a fragment of the human dysferrin protein, wherein the polynucleotide comprises:
i. the nucleotide sequence of SEQ ID NO: 2 or 8;
ii. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of SEQ ID NO: 2 or 8 over the entire length of SEQ ID NO: 2 or 8 identical nucleotide sequences;
iii. the nucleotide sequence of SEQ ID NO: 14 or 16;
iv. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of SEQ ID NO: 14 or 16 over the entire length of SEQ ID NO: 14 or 16 identical nucleotide sequences;
v. A polynucleotide encoding a fragment of hDYSF protein, wherein the fragment of hDYSF protein consists of the amino acid sequence of SEQ ID NO: 10; or
vi. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical polynucleotides;
made up of; and
(c) a second ITR;
Including,
wherein the polynucleotide is flanked by the first and second ITRs. 73. The method of claim 72,
An AAV vector, further comprising one or more polynucleotides selected from a selectable marker, an origin of replication (ORI), an untranslated region (UTR), or a polyadenylation (polyA) signal. The method of claim 72 or 73,
wherein the ITR is an AAV ITR. 75. The method of claim 74,
wherein the AAV ITR is an AAV2 ITR or an AAV3 ITR. 76. The method of any one of claims 72 to 75,
wherein the ITR comprises the nucleotide sequence of SEQ ID NO: 3 or 17. The method of any one of claims 73 to 76,
The polyA signal is an artificial polyA signal, AAV vector. 78. The method of any one of claims 73 to 77,
The polyA signal comprises the nucleotide sequence of SEQ ID NO: 7, AAV vector. 79. The method of any one of claims 73 to 78,
AAV vector, wherein the selectable marker is an antibiotic resistance gene. 80. The method of claim 79,
The antibiotic resistance gene is a β-lactamase gene or a kanamycin resistance gene, AAV vector. The method of any one of claims 72 to 80,
The AAV vector comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 16. The method of any one of claims 72 to 81,
Wherein the AAV vector does not further comprise a second polynucleotide sequence encoding a second fragment of the hDYSF protein. The method of any one of claims 72 to 82,
wherein the AAV vector does not contain AAV sequences other than one or more ITRs. The method of any one of claims 72 to 82,
wherein the AAV vector does not contain viral sequences other than one or more ITRs. A dual adeno-associated virus (AAV) vector system comprising:
(a) a first AAV vector, wherein the first AAV vector comprises the AAV vector of any one of claims 40-42 and 49-71; and
(b) a second AAV vector, wherein the second AAV vector comprises the AAV vector of any one of claims 43-45, claims 72-84;
A double adeno-associated virus (AAV) vector system comprising a. 86. The method of claim 85,
The AAV vectors are AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV- 12, AAV-13, AAVrh.10, AAVrh.20, or AAVrh.74, a dual adeno-associated virus (AAV) vector system. 86. The method of claim 85,
The AAV vector is AAVrh.74, a dual adeno-associated virus (AAV) vector system. An adeno-associated virus (AAV) packaging system comprising:
(a) a plasmid comprising the recombinant polynucleotide of any one of claims 1 to 38;
(b) an adenovirus helper plasmid; and
(c) rep-cap plasmid;
, Adeno-associated virus (AAV) packaging system comprising a. An adeno-associated virus packaging system comprising:
(a) a plasmid comprising the recombinant polynucleotide of any one of claims 1 to 38; and
(b) an adenovirus helper plasmid;
Including, adeno-associated virus packaging system. The method of claim 88 or 89,
The adeno-associated virus packaging system of claim 1, wherein the adenovirus helper plasmid comprises a pHELP plasmid. A method for producing an adeno-associated virus (AAV) vector,
89. The method comprising contacting a cell with a plasmid comprising the recombinant polynucleotide of any one of claims 1-38, or the AAV packaging system of claim 88. 92. The method of claim 91,
wherein the cell is a host cell, optionally a mammalian host cell, and further optionally a HEK293. A method for producing an adeno-associated virus (AAV) vector,
The method comprises transducing a packaging cell line with a plasmid comprising the recombinant polynucleotide of any one of claims 1 to 38, wherein the packaging cell line rep. adeno-associated virus And to express the cap gene, the method. 94. The method of claim 93,
Wherein the AAV rep gene is Rep78. 94. The method of claim 93,
Wherein the AAV cap gene is a Rh74 cap gene. A cell comprising the recombinant polynucleotide of any one of claims 1-38. A cell comprising a plasmid comprising the recombinant polynucleotide of any one of claims 1 to 38. The method of any one of claims 88 to 97,
wherein the plasmid comprises a polynucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18 or 19. , method, or cell. The method of any one of claims 88 to 97,
wherein the plasmid comprises a polynucleotide of SEQ ID NO: 18 or 19, an AAV packaging system, method, or cell. As a method of treating dysferlinopathy,
The method is directed to a subject in need thereof:
(a) an effective amount of a first polynucleotide, wherein the first polynucleotide is the recombinant polynucleotide of any one of claims 1-24; and
(b) an effective amount of a second polynucleotide, wherein the second polynucleotide is the recombinant polynucleotide of any one of claims 25-38;
A method comprising administering a 101. The method of claim 100,
wherein the first polynucleotide and/or the second polynucleotide is administered intramuscularly or intravenously. 101. The method of claim 100 or 101,
wherein the first and second polynucleotides are administered simultaneously or sequentially. As a method of treating dysferinopathy,
The method is directed to a subject in need thereof:
(a) an effective amount of a first adeno-associated virus (AAV) vector, wherein the first AAV vector is the AAV vector of any one of claims 40-42 and 49-71; and
(b) an effective amount of a second AAV vector, wherein the second AAV vector is the AAV vector of any one of claims 43-45 and 72-84;
A method comprising administering. 104. The method of claim 103,
wherein the first AAV vector and/or the second AAV vector is administered intramuscularly or intravenously. 104. The method of claim 103 or 104,
wherein the first and second AAV vectors are administered simultaneously or sequentially. As a method of treating dysferinopathy,
88. A method comprising administering to a subject in need thereof an effective amount of the AAV dual vector system of any one of claims 39 or 85-87. 107. The method of claim 106,
wherein the AAV dual vector system is administered intramuscularly or intravenously. As a method of treating dysferinopathy,
49. A method comprising administering to a subject in need thereof an effective amount of the composition of any one of claims 46-48. 108. The method of claim 108,
wherein the composition is administered intramuscularly or intravenously. According to any one of claims 100 to 109,
The method of claim 1, wherein the dysperinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy. Use of the composition in the manufacture of a medicament for the treatment of dysferinopathy in a subject in need thereof, the composition comprising:
(a) a first polynucleotide, wherein the first polynucleotide is the recombinant polynucleotide of any one of claims 1-24; and
(b) a second polynucleotide, wherein the second polynucleotide is the recombinant polynucleotide of any one of claims 25-38;
Including, uses. Use of the composition in the manufacture of a medicament for the treatment of dysferinopathy in a subject in need thereof, the composition comprising:
(a) an effective amount of a first adeno-associated virus (AAV) vector, wherein the first AAV vector is the AAV vector of any one of claims 40-42 and 49-71; and
(b) an effective amount of a second AAV vector, wherein the second AAV vector is the AAV vector of any one of claims 43-45 and 72-84;
Including, uses. A use of the composition in the manufacture of a medicament for the treatment of dysferinopathy in a subject in need thereof, wherein the composition comprises the AAV dual of any one of claims 39 or 85-87. Uses, including vector systems. Use of the composition of any one of claims 46 to 48 in the manufacture of a medicament for the treatment of dysferinopathy in a subject in need thereof. The method of any one of claims 111 to 114,
Wherein the composition is administered intramuscularly or intravenously. The method of any one of claims 111 to 114,
Wherein the dysperinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy. According to any one of claims 100 to 110,
An effective amount of the first AAV vector is about 1×10 ⁶ to 1×10 ¹⁶ vg/kg, about 1×10 ⁸ to 1×10 ¹⁵ vg/kg, or about 1×10 ¹⁰ to 1×10 ¹⁴ vg/kg. wherein the dose is determined by a supercoiled PCR quantification standard. 117. The method of claim 117,
wherein the effective amount of the first AAV vector is 1.2e13 vg/kg, wherein the dosage is determined by a supercoil PCR quantification standard. The method of any one of claims 100 to 110 and 117,
An effective amount of the second AAV vector is about 1×10 ⁶ to 1×10 ¹⁶ vg/kg, about 1×10 ⁸ to 1×10 ¹⁵ vg/kg, or about 1×10 ¹⁰ to 1×10 ¹⁴ vg/kg. Wherein the dose is determined by a supercoiled PCR standard. 119. The method of claim 119,
wherein the effective amount of the second AAV vector is 1.2e13 vg/kg, wherein the dosage is determined by a supercoil PCR quantification standard. The method of any one of claims 117 to 120,
wherein the total dose of the AAV vector is 2.4e13 vg/kg, wherein the dose is determined by supercoil PCR standards. As a recombinant polynucleotide encoding human dysferrin (hDYSF) protein,
The recombinant polynucleotide sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide sequence of SEQ ID NO: 20 or the nucleotide sequence of SEQ ID NO: 20 A recombinant polynucleotide comprising % identical nucleotide sequences. 122. The method of claim 122,
The recombinant polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 20. A method for producing the recombinant polynucleotide of claim 122,
A method comprising contacting a cell with the recombinant polynucleotide of any one of claims 1 - 24 and the recombinant polynucleotide of any one of claims 25 - 38 . A method for producing the recombinant polynucleotide of claim 122,
A method comprising contacting a cell with the dual AAV vector system of any one of claims 39 and 85-87. 125. The method of claim 124 or 125,
The method of claim 1, wherein the cell is a eukaryotic cell. 125. The method of claim 124 or 125,
wherein the cells are muscle cells, cardiac cells, stem cells, satellite cells, and/or liver cells. A method for producing the recombinant polynucleotide of claim 122,
A method comprising administering to a subject the recombinant polynucleotide of any one of claims 1 and 24 and the recombinant polynucleotide of any one of claims 25 and 38. A method for producing the recombinant polynucleotide of claim 122,
A method comprising administering to a subject the dual AAV vector system of any one of claims 39 and 85-87 or the composition of claims 47 or 48. As a method of treating muscular dystrophy in a subject,
A method comprising expressing the recombinant polynucleotide of claim 122 in the subject. 130. The method of claim 130,
A method comprising administering to a subject the recombinant polynucleotide of any one of claims 1 to 24 and the recombinant polynucleotide of any one of claims 25 to 38 or the composition of claims 47 or 48. 130. The method of claim 130,
88. A method comprising administering to a subject the dual AAV vector system of any one of claims 39 and 85-87. The method of any one of claims 128 to 132,
The subject is human, non-human primate, canine, ovine, horse, porcine, murine, rat, rabbit, bovine, or a mammal selected from a feline. The method of any one of claims 128 to 133,
Wherein the subject is suffering from dysperinopathies. 134. The method of claim 134,
The method of claim 1, wherein the dysperinopathy is limb muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy.

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